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Werner Kollath, German bacteriologist
Probleme & praktische Lösungen
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Gel Electrophoresis Size Marker
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Wichtiges Wissenswertes Wunderbares aus Chemie & Biologie
We offer our customers an extensive library, with numerous oving quality o Impr
Nukleinsäure-Dekontamina mit der ExitusPlus™-Tec tion hnologie
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Die moderne Gentechnik zeigt, dass in vielen Fällen schon freie DNA-Moleküle für Infektionen, Rekombin ationen oder biologische Transformationen ausreiche n [1,2]. Zusätzlich werden die Nachweisverfahren für DNA-Moleküle immer sensitiver. Daher wird die Detektion von Kontaminationen oder die Verhinderung von Amplifikations-Artefakten in der PCR für die Gentechn ik, die Kriminalistik, die Biomedizin und die Hygiene immer wichtiger. Die vollständi ge Dekontamination von Geräten und Materialie n von DNA-Molekülen wird so zu einem entscheidenden Faktor für die allgemeine biologische Sicherheit.
Using ready-to-use ELISA
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Keywords NukleinsäureDekontamination DNA-Degradationstest PCR-Test
A comprehensive catalogue of products, with detailed
Antibody Stabilisation Autoklavieren von DNA ELISA Plates Cross-reactivity Interfering effects
information provided by no other catalogue in the field, is available in German and English.
Alles oder Nichts: Erstaunlich e Erkenntnis Das Mittel der Wahl zur Beseitigung von Kontamina Kontaminationen durch Nukleinsäuren ist immer noch Chlorbleichlauge („bleach“) – ein Mittel das alles zerstört, nicht nur die Nukleinsäure. Dies hat uns veranlasst in Kooperation mit multiBIND Biotech, Köln, nach einer unschädlichen Alternative zu suchen und die molekulare Wirkungsweise sonstigen DNA-Dekontaminationsmittel der auf dem Markt befindlichen zu untersuchen. Hierfür wurde unter suchen. sehr hoher Belastung (großer DNA-Überschuss) mit definierten DNA-Kontami DNA-Über nationen die Eigenschaften der konventionelle n Mittel verglichen. Zwei Probleme werden offensichtlich: Erstens werden durch die konventionellen Mittel in keinem Fall die DNA-Moleküle effizient zerstört und zweitens enthalten diese Mittel Komponenten mit stark korrosiven oder giftigen Eigenschaften für uns die Notwendigkeit der Neuentwicklun . Als Fazit daraus hat sich g einer effektiven Lösung zur DNA-Dekontam DNA-ExitusPlus™ und Autoclave-Exit ination ergeben, die wir hier als usPlus™ vorstellen. Im Vergleich zu den herkömmlichen Produkten wird RNA schnell und effizient zerstört, ohne DNA und dass das Reagenz korrosive oder giftige Eigenschaften aufweist. Bei der DNA-Dekontamination unterscheidet man nach der molekularen Wirkungsweise der eingesetzten Mittel drei Grundprinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Modifika Je nach Zusammensetzung der Mittel Denaturierung und Degradation. können diese drei Prinzipien einzeln oder in Kombination angewandt werden. Da nach den aktuellen Erkenntnissen zum biologischen Risikopotenzial von freien DNA-Molekülen für eine wirklich DNA-Dekontamination die Zerlegung sichere dieser DNA-Moleküle in möglichst kleine Fragmente die wirkungsvollste Methode wurden die gängigen konventionellen tionellen Mittel mit unserer Neuentwicklun ist, g DNA-ExitusPlus™ im DNA-Degradat glichen. Der DNA-Degradationstest erlaubt ionstest verver einen sensitiven, quantitativen Vergleich der Geschwindigkeit des DNA-Abbaus (Abb. 1 und 2). Unerwarteter Weise haben wir festgestellt, dass einige der bekannten kommerziellen Mittel nur mit dem Prinzip der Modifikation oder Denaturierung der DNA-Moleküle arbeiten. Eine Zerlegung der DNA-Stränge genetische Information, für die diese erfolgt dabei nicht, sondern die Informa DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine chemische Demaskierung der DNA-Moleküle durch Entfernung der blockierenden Gruppen würde die genetische Information wieder lesbar plifizierbar machen. Nach dem heutigen und amWissensstand zur Gentechnik und der Problematik der Neukombination von trägern sind solche Mittel eigentlich nicht Erbmehr zeitgemäß. Aber auch die Mittel, die zu einer nachweisbaren Degradation
Why is there such a great difference in storage between h The reason is that in professional ELISA kit production the plates are not easy to perform process has been an industry standard for thirty years. Fo coating stabiliser solution. It is just as simple as a “second blocking step lutions freely available in low volumes for use in research lab until now. AppliC in volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabi to-use and has a great advantage compared to almost any stabiliser used antibodies and antigens than most other products do. And there is a secon Two benefits with one solution When antibodies are coated onto ELISA plates, most of the antibodies are not into close contact to the plastics surface of the ELISA plate, conformational ch The result is that most antibodies coated on a plate are unfolded or inactive. O active and can bind to analytes and this is greatly variable depending on the s really differ from batch to batch or even from well to well. These differences from well to well can affect the variability of an assay, bec way of refolding antibodies and of preserving antibodies from conformati decrease such variabilities in assay performance. This is a key benefit of Ap coated proteins to refold and then to preserve active conformation over a lo antibody conformation of some of the coated antibodies and 2. Preserving c fits are used for production of high-quality ELISA kits as well as in resear Stabiliser the percentage of active antibodies will still be in the range of 2–8 from well to well and from plate to plate can be minimised in most assays depend on the used antibodies, but when ELISA are validated (e.g. according Validation”, FDA, 2001) or according to other validation strategies, the differe The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwi
Dekontamination der Haut und Hände von Nukleinsäure n
AppliChem brings advantages through knowledge.
Freie Nukleinsäuren verursach en als Kontaminationen große Probleme im Forschungs- und molekular biologisch-analytischen oder klinisch-diagnostischen Labor. Durch die extrem hohe Sensitivität von DNA-Nach weistests, können kleinste Verunreinigungen in PCR-Ansä tzen zusätzliche Arbeit bedeuten und im schlimmsten Fall Ergebnisse verfälsch en. Mit Derma-ExitusPlus™ (HHDK) aus der Serie von ExitusPlus™-Produkten wird erstmals ein völlig neuer Anwendungsbereich erschlossen bzw. zusätzlich e Kontaminationsquellen ausgeschlossen.
Size-Exclusion Chromato grap for purification of biomolec hy ules
Size-exclusion chromatog raphy (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significant ly slower through the column than larger molecules. Not to be mixed up with gel electropho resis, there are big difference s in terms of the separation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.
DNA-freie Reagenzien und Mastermixe für die PCR
Der Anteil von molekular biologischen Nachweismethoden ist in den letzten Jahren erheblich gestiegen, besonder s in den Bereichen Qualitätsk ontrolle, Forensik, klinischer Forschung und Diagnostik – insbesondere der Infektionsdiagnostik . Gerade für diese Applikatio nen werden hochsensitive und gleichzeitig zuverläss ige PCR-Tests benötigt. Dafür bietet AppliChem nun optimiert e PCR-Kits an und widmet sich explizit der Hintergru ndproblematik, die durch DNA belastete Reagenzie n und Arbeitsplätze entstehen kann.
ine der Hauptquellen für Kontamination en mit Nukleinsäuren ist der Experimentato r selbst. Die Nukleinsäuren stammen B. aus Hautschuppen, Haaren und Speichel oder von Mikroorganismen, die seine Haut besiedeln oder z.B. beim Niesen eigesetzt werden. Gelangen diese in die PCR-Ansätze oder PCR-Reagenzi en, können s
It is indeed correct that smaller molecules
pass more slowly through t
Limiting Cross Reactivity in Immunoassays Without Blocking .... No Result! Tips for storing antibodies Comparing Blocking Reagents Improving quality & stability of ELISA Products with application notes Antibody Stabilizer-PBS Antibody Stabilizer-Tris Applicoat Plate Stabilizer Blocking Buffer I Blocking Buffer II EGrade Blocking Buffer III BSA Blocking Reagent CA Coating Buffer pH 9,6 Coating Buffer pH 7,4 CrossDown Buffer CrossDown Peroxidase-Stabilizer Peroxidase-Stabilizer Sample Buffer TSample Buffer T+ Stripping Buffer I Washing Buffer TrisT- (10X) Washing Buffer TrisT+ (10X) Related products Further reading 21 21 22 23 24 25 26 27 27 28 31 32 33 33 34 34 35 36 36 2 10 12 14 18
© 2010 AppliChem • Immunoassay Buffer
Limiting Cross Reactivity in Immunoassays
Antibodies are used in Immunoassays to easily and specifically distinguish between different substances.
Dr. Wolfram H. Marx, AppliChem; PD Dr. Wiesmann, University of Münster; Dipl. Biol. Susanne Siewert, University of Ulm; Dipl. Chem. Nico Dankbar, University of Münster; Dr. Peter Rauch, CANDOR Biosciennce GmbH; Dr. Christoph Specht PARA, Bioscience GmbH There are several types of these assays including Enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Western blotting, radioactive labelled immunoassays (RIA), protein arrays, immuno histochemistry, and immunopolymerasechain reaction (ImmunoPCR). Each of these assays have one drawback in common cross reactivity. Immunoassays are a very important tool in bioanaly tical and biochemical laboratories. They are used in research, food and environmental monitoring as well as in diagnostic applications. Immunoassays are quite easy to carry out and very specific in terms of quanti tative and qualitative significance due to their use of antibodies for detection. Theoretically, each antibody can identify one antigen and binds this antigen with high affinity which explains why one can distinguish so easily between different substances. In practice, it is not that simple. Immunoassays suffer from cross reactivity which results in false bands in Western blots, signals in the negative control of an ELISA or a very high background in a protein array. Every false result means more work, additional costs and potentially misdiagnosis of patients . Although antibodies are very specific and have high affinity for one antigen in particular, often antibodies can also bind with lower affinity to other antigens which are not detected by the assay. This is even observed with very
labeled detection antibody analyte capture antibody heterophilic antibodies or HAMA blocking protein of surface interfering or cross reacting substance
Fig. 1 The perfection: interference-free sandwich assay. Fig. 2 A on-specific binding of a labeled detection N antibody to a not (sufficiently) blocked surface. Result: false-positive signal. B on-specific binding of a labeled detection N a ntibody to a blocked surface. Despite blocking of the surface the antibody binds to the blocking protein itself. Result: false-positive signal. C n interfering protein binds to the Fc segment A of the detection antibody and hinders sterically the binding of the analyte. Result: false-negative signal. D he capture antibody binds to the Fc segment of T the labeled detection antibody. The analyte can- not be bound by the capture antibody any more. Result: false-positive signals
Immunoassay Buffer • AppliChem © 2010
to foreign proteins of the real sample (fig. In protein arrays this phenomenon leads to higher background fluorescence of single spots or a low signaltonoise Interferences in immunoassays Interference can come in several forms such as cross reactivities. while the secondary labelled antibody. nonspecific binding and matrix effects. most of the above men tioned effects can be avoided. Assays with insufficient blocking or with difficult matrices con taining e. 2C) or to the capture antibody (fig. To understand the basic principles of interference. Since detection antibodies are labelled with enzymes (e. The capture antibody binds the analyte. 1) followed by fig. a high albumin concentration or high con centration of endogenous interfering substances. resulting in no binding Result: false-positive signals. J ross reactivity of an interfering substance with C the detection antibody. Similarly. which are frequently hydrophobic. nonspecific binding to albumin or immunoglobulins). antibody.g. The antigenantibody bin ding can be impaired too . binding to surfaces (e. fluorescent dyes. Laboratory or clinical samples may contain foreign sub stances in concentrations that can interact with the ana lyte or the capture/detection antibodies thereby dis rupting the desired reaction. There are also other causes of non specific binding. The rest of the surface is blocked sufficiently. The interfering antibody binds in the Result: false-negative signal. cross reactivities and matrix effects leading to high background with bad signaltonoise ratio. The interfering antibody binds in the area of the highly variable region of the Fab segment and prevents the binding of the analyte. Result: false-positive signals Such a phenomenon is rather seldom in practice. CrossDown Buffer). alkaline Phosphatase or Peroxidase). 3 I ross reactivity of an interfering substance with C the capture antibody. 2–5 where interference is demon strated. Result: false-negative signal. In a troublefree sandwich ELISA. 4 Fig. The epitope is blocked for binding capture and detection antibody. respectively. area of the highly variable region of the Fab segment and thus prevents the binding of the analyte. © 2010 AppliChem • Immunoassay Buffer 3 . Fig. the label itself can also be a source of unwanted interactions. H n anti-idiotypic HAMA binding to the detection A antibody. binds to a different site on the analyte (fig. Fig. 2A and 2B). surfaces that act as platforms for immunoassays have also been known to be a source of interference. Western blotting membranes or ELISA wells). Result: false-negative signal. Non-specific binding Nonspecific binding occurs when an antibody binds to substances present in much higher concentrations than the target analyte (e. are strongly affected. The dyes themselves may cause unwanted binding and thereby reduce the solu bility of the labelled protein. we first take a look at an ideal sandwich ELISA (fig.g.g. 5 F ridging by HAMAs and heterophilic antibodies.well characterised antibodies known to have a high affinity to the target analyte. or binding to loci on immobilized antibodies in protein arrays . In those cases false positive results are obtai ned even in the absence of an analyte or the whole assay shows a high background.g. Result: false-negative signal. the capture antibody is immobilised on the bottom of a well. Such an interference picture may occur with antibodies directed to a target with a conserved amino acid sequence of a protein whose sequence motive also occurs in other proteins. These effects may lead to increased nonspecific binding of the labelled antibody onto surfaces (fig. K ross reactivity both with the capture and with C the detection antibody. 2D). resulting in a coupling of the specimen. radioactive isotopes or DNA (Immuno PCR). L asking of the analyte by a protein of the B M respectively. the binding properties of detection anti bodies can be changed. 1). By applying novel buf fers (e. In the case of fluorescent dyes. Simply exchange the sample buffer or antibody dilution buffer by CrossDown Buffer and thereby improve the quality of the assays and the efficiency of the assay development. The result are interferences such as nonspecific binding. but definitely possible with antibodies having lower specificity.g. to the analyte at all or in the case of a sterical G n anti-idiotypic HAMA binding to the capture A hindrance binding is very week. of the capture antibody. Result: false-negative signal.
There is a smooth tran sition to all other negative effects. inter ferences and false results are severe. In sandwich ELISAs based on monoclonal mouse antibodies. Unlike nonspecific binding. sometimes the additional bands simply represent protein fragments which originate from the “normal” degradation pro cess. 4]. Since the results of these assays are the basis of patient therapies. Fluorescent dyes also may bind pro teins or antibodies from serum samples. There are negative effects that are restricted to medical and diagnostics assays. IgM or IgE type and are formed as an immune response after contact with animal immunglobulins. Anti-Animal-Antibodies Human AntiAnimalAntibodies (HAAA) are of the IgG. HAMAs interfere with immunological methods which include mouse antibodies. 3J and 3K).g. Based on this. 4F). these structures are similar to the analyte such as metabolites or chemical substances with a similar molecular structure.immunoassay ratio. HAMAs are human antibodies which are relatively specific and which can bind mouse antibodies with a middle affinity up to. heterophilic anti bodies. The complexity of protein arrays is very high due to the application of many different capture antibodies and labelled detec tion antibodies in one reaction. respectively. These effects are based on interfering substances present in human specimen like plasma. IgA. serum or tissue samples. Cross reactivities play a key role in many competitive assays. Thereby. which can affect the determina tion of the target analyte . For these assays. because only one antibody is used [1. it is part of the validation to identify and to quantify possible cross reactors experimentally . Often. in some rare cases. Matrix effects can be caused by AntiAnimalAntibodies. capture and detection antibody will be bridged (fig. speaks about a matrix effect. endogenous interfering substances or influ ences like viscosity. 3I. If the exact molecular cause of such an effect is unknown. The concentrations can be very high. Matrix effects The least defined term is the “matrix effect”. resulting in a reduction of the dye fluorescence and in extreme cases to a complete quenching of the signal. But in some cases it is important to look closer at cross reactivities caused by the primary or secondary antibody. the risk of nonspecific binding of proteins in the sample or labelled antibodies to single spots increases significantly as well as interferences of components of the sample with the antibodies . For any scientific publication it is necessary to verify the reasons for unexpected bands and signals anyway. After thera peutic medication the human immune system reacts to these foreign antibodies and begins to produce anti bodies against these mouse antibodies. Proteins with evolutional homology of amino acid sequence or similarity in tertiary structure can cross react too. resulting in a false Cross reactivity Cross reactivities are the result of nontarget binding and appears similar to nonspecific binding. Cross reactivity is the ability of the antibody to bind structures other than the target analyte (fig. a high affinity. without knowing the exact molecular reasons for this unwanted binding. Studies report that up to 80 % of all samples contain HAAAs. HAAAs are well known from diagnostic assays. In Western blots. however. pH or salt concentration. Therefore.g. discussions are ongoing to eliminate fluorescent dyes from protein arrays completely . in cancer therapies). reaching levels of several milligrams per milliliter . HumanAntiMouse Antibodies (HAMA) are the best known interfering antibodies in immunoassays. by measurement of the competing concentration of the cross reacting species . Cross reactivities can also play a major role in the detection of proteins in Western blots or in immuno histochemical applications. Matrix effects are the sum of all negative effects of all compo nents within a sample. One of the reasons for the development of these antibodies can be nonhuman therapeutic antibodies which are admin istered as drugs (e. but can be related to the composition of the sample to be determined. Cross reactivity can result in staining of additional bands in Westerns or cell struc tures. when one talks about cross reactivity the cross reacting substance is known and its cross reacting properties can be proven e. one 4 Immunoassay Buffer • AppliChem © 2010 .
while others bind to the Fabfragment resulting in redu ced binding of the analyte or even completely preven ting the formation of any real complexes. Rheumatic sera lead to a linkage of capture and detection antibo dies with the consequence of falsepositive signals. The binding ability of certain proteins is a sub stantial part of their biological function. Some well known interfering substances in human sera are albumins. Numerous hormones are bound to transport proteins. albumin. 2C. this reduces the accessibility of the anti body to the analyte. Since analytes of low molecular weight can bind readily to albumin. Endogenous proteins can bind as interfering factors to antibodies (fig. Rheumatic factors are IgM type antibodies which bind to Fcfragments of human antibodies. antiidio typical interfering antibodies bind the highly variable. Contact with domestic animals over several years enhance the formation of anti animal antibodies that either bind to antibodies of a single species (e. 5L). Due to the sequence homology between antibodies of different species. due to the low concentration of the analytes. 'heterophilic antibodies are antibodies which bind other antigens than the specific antigen'. Some interfering antibodies can bind to the Fc portion of the antibody. Drugs aren't the only reason for the development of HAMAs. the interfering antibodies are weakly binding antibodies . nonspecific binding or even cross reactivity is possible which complicates the recognition of certain analytes in an assay similar to antibodies. Heterophilic antibodies According to Taber’s Medical Dictionary. which predominantly disturb assays that. hamster) or binding to antibodies of several species with different affinities. The difference between HAAAs and heterophilic antibodies is their formation. IgM. Fab portion of the antibody . e. mouse. but rather they are multispecific anti bodies of the early immune response or interfering antibodies with unknown immunological origin . The latter aren't formed upon contact with animal immunglobulins. and therefore they may bind to the Fcfragments of antibo dies of other species in the assays as well. IgA or IgE type. The conse quence is a false negative measurement (fig. Because these proteins are natural receptors for many sub stances. which may lead to difficulties as well.g.g. In contrast. 4G and 4H). antibody fragments or high concen trations of animal immunoglobulins. Heterophilic antibodies can be of the IgG. In general. For example. The effect of the rheumatic sera is similar to the effect of HAAAs. rabbit. These sera do contain so called rheumatic factors in high concen tration. Interference caused by endogenous components of the specimen Even naturally occurring proteins found in specimens can interfere with immunoassays. Interference by HAAAs or by heterophilic anti bodies have been known now for more than 30 years. require a low dilution of serum or plasma specimens . are able to reduce the negative effects of the HAAAs or heterophilic antibo dies by competition. The IgM type plays a key role in sera from rheumatic patients. antibodies which have © 2010 AppliChem • Immunoassay Buffer 5 . HAMAcontaining sera may disturb assays which contain antibodies from other species. The ability of HAAAs to bind the Fcfragment is called antiisotypical interference. but don’t always prevent them . 3IK) or mask the target analyte (fig. Therefore. lysozymes and fibrinogen . Addi tion of blocking substances to the sample buffer. nonspecific sera. This reflects the general interfering mechanism of hete rophilic antibodies. e.positive signal. lysozyme binds nonspecifically to any proteins with a low isoelectric point. complement and Creactive protein (CRP). complement factors. dog.g.
a chemical modification during the manufacturing process allows the produc tion of casein solutions with reproducible and reliable results. The purified antibodies were immobilized on aminosilane functionalized microarray slides using a spotter (GMS 417) at a concentration of 500 µg/ml in a volume of 1.3. Originally. An example of the impact of a matrix effect on an ELISA is shown in figure 9. With this model assay a 6 Immunoassay Buffer • AppliChem © 2010 . was used to develop a new buffer. The staining of the cultures intensifyes with time (fig. the easier is the blocking. low to medium affinity binding. The larger the analyte. CrossDown Buffer reduced a high background and improved the signaltonoise ratio from 3. Figures 7 to 10 show different examples of typical in terference effects in immunoassays that are prevented by the use of CrossDown Buffer. The best known strategy to circumvent the negative effects is an optimised blocking procedure. One other important aspect. That kind of casein blocker is available from many suppliers. To get the systems run ning many blocking solution were developed.4 to 17. 6 upper right). The expected increase in osteocalcin expression can be correctly monitored by using Blocking Buffer I. By applying a novel casein based blocking reagent (Blocking Buffer I. 6 lower left). Figure 7 shows a Western blot with high back ground. Small analytes often require a more efficient blocking. Positive results in a protein chip application are shown in fig. TaKaRa). Caseinbased blocking solutions have proven to be very efficient. Applying the conventional protocol. The solution to combating the one common feature of many interfering substances. a significant reduction of back ground is achieved (fig. allowing the selection of antibodies in terms of their suitability to detect EPIL. Replacement of an unsuitable blocking reagent in immunohistochemistry makes the interpretation of an experiment with osteoblast culture possible (fig. It elimi nates low and medium affinity binding. the problems in many immunoassays are caused by low to medium affinity bindings.8 nl/spot. In many cases the substitution or optimisation of the blocking reagent alone is not sufficient. Standard blocking with BSA leads to a completely falsepositive result (fig. Only such a multipurpose solution can help saving time and money for optimizing and developing immunoassays. After washing of the slides they were analyzed with a flu orescence scanner (GMS 418) and the data were evalu ated with ImaGene (Biodiscovery Inc. The optimal blocking buffer shall be a generally applicable solution for most immunoassays and shall give reliable results. because some analytes are fatsoluble and the binding between antibody and analyte can be affected by lipids. 2 ml supernatant of an EPILover expressing cell line (SKBR3) were mixed with the dye Oyster650P (Denovo Biolabels) and all proteins of the mixture were labeled. The incubation on the slide was carried out at a dilution of the medium : buffer 1 : 20 with CrossDown Buffer in comparison to PBS.3 % BSA in TBS was applied and detection was done with ECL (Amersham). if it is cut into fragments of different mole cular weights. i. With the substitution of Blocking Buffer I and CrossDown Buffer as new anti body dilution buffer.). The use of CrossDown Buffer resulted in a clear reduction of the background signal. 8. the actual expression is correctly detected (fig. The reason is simple. Only the substitution of the blocking reagent and additionally substitution of the antibody dilution buffer led to an analyzable result: Myostatin (GDF8. CrossDown Buffer (AppliChem) capitalizes on the fact that the binding of interfering substances is weaker than the specific binding of the target analytes. is the interference by strongly fatty speci mens. Avoiding the interference by applying novel immunoassay buffers – examples from the practice In most cases. Simply solubilizing casein doesn’t give a good blocking solution. With time. blocking was perfor med with 2 % nonfat dried milk powder and 1 % BSA in TBS. AppliChem) in combination with antiosteocalcin (monoclonal. 6 upper left). but rather lack real good results in practice. but the results in assays are not of the same quality like they should be.immunoassay an isoelectric point of approximately 5. because the blocking agent has limited influence on all the different negative effects. But preparing such a solution with consistent blocking efficiency requires a great deal of time and experience. can be bound and form a bridge between capture and detection anti body . Afterwards. Literature describes and practice shows that casein works best. 6). a freshly prepared osteoblast culture shows no or a very weak expression of the extracellular matrix protein osteocalcin. Nowadays. In this experiment different polyclonal anti EPIL antibodies (EPIL – early placenta insulin like growth factors) were tested for their suitability. One of the typical daily problems in many labs. On the first day. the cultured osteoblasts build up the extra cellular matrix and osteocalcin is synthesized. This holds true especially if expensive antibodies or difficulttoprepare samples are applied. 12 kDa) from mouse myoblasts (C2C12) was blotted on nitrocellulose NC45 (Serva) and detected with anti GDF8 (Santa Cruz). the bands are hardly visible. 7).e. which should be mentioned. As antibody dilution buffer 0. Most of them can be called 'very creative'. without nega tively affecting high affinity binding and high specificity.
3. Chem. Siewert. Whereas blocking with BSA gives a wrong result (upper right). Germany) © 2010 AppliChem • Immunoassay Buffer 7 . Dankbar. Biol. S. Wiesmann. University of Ulm.4 to 17. 6 Osteoblast culture for detection of osteocalcin. 7 Western blot. Detection of myostatin in mouse myoblasts with anti-GDF-8 as primary and rabbit anti-goat IgG-HRP as secondary antibody on a nitrocellulose membrane NC45. (Dipl. Signal-to-noise could be increased from 3. AppliChem) shows correct results (upper left). (Dipl. N. The time course of expression can be shown correctly (lower panels). 8 Reduction of a non-specific interaction of the detection antibody with the array surface by the use of CrossDown Buffer. (images by PD Dr. University of Münster. Germany) Fig.Fig. University of Münster. Germany) with Standard Assay Buffer with CrossDown Buffer Fig. switching to a novel caseinb ased blocker (Blocking Buffer I. Left side without and right side with Blocking Buffer I and CrossDown Buffer.
05 µg/ml in PBSBSA buffer) with ImmunoPure®TMBsubstrate (Pierce). It leads to a better sensitivity by reducing the level of detection (LOD) from 0. 10 is an example. The improvement can be explained by the elimination of the falsepositive signals of the preimmun sera and the reduction of the background. the biotinylated antibody from Clone C6 (Biotrend. The result of using the standard buffer (PBS/NaCl/Tween® 20) in contrast to the new CrossDown Buffer is selfevident. The spiked serum samples were diluted either with a PBS BSA buffer or with CrossDown Buffer 1 : 2 and mea sured by ELISA.051 to 0. a company. which develops and validates assays for pharmaceu tical research and diagnostic applications. CrossDown Buffer pre vented the binding of CRP to endogenous substances of the rabbit serum and thus improved the sensitivity of the calibration curve by the factor of 3 (fig. Detection was carried out with NeutrA vidin™ – conjugated horseradish peroxidase (Pierce. where the substitution of sample and antibody dilution buffer by CrossDown Buffer was sufficient to achieve a good result. Blank values are in column 5. The assay was performed by Candor Bioscience GmbH. Rauch. Extinction 450 nm •PBS/BSA-Standard buffer CrossDown Buffer CRP [ng/ml] Fig. A matrix effect. 1 µg/ml coating concentration in PBS) and for detection.immunoassay matrix effect was induced systematically. whose exact molecular reason is not known.065 and enlarging the measurement range. CrossDown Buffer improved sensitivity by avoiding a matrix effect. As antigen a lysate of human kidney carcinoma cells were immobilized and a serial dilution of two im mun sera in repeat determination (1:50 to 1:36450) A–G loaded in columns 1–4.152 to 0. probably causing a significant reduction in the accessability of the epitope by the antibody. 9). Clone C2 was used (Biotrend. Candor Bioscience GmbH). working concentration 2 µg/ml) was applied.022 and the level of quantification from 0. As capture antibody. leads to a calibration curve with low sensi tivity. 8 Immunoassay Buffer • AppliChem © 2010 . The corresponding preimmun sera (1 : 50) were pipetted in lane H. CRP is able to bind many proteins and substances (scavenger func tion of CRP). although interfering effects as shown in figure 3I K can not be excluded. working concentration 0. Rabbit serum was used as a matrix and spiked in defined concen trations with human Creactiveprotein (CRP. an interfering effect as shown in figure 5L takes place. Biotrend). Pre sumably. Due to its physiological function. 9 ELISA of CRP in rabbit serum (developed by P. Again. The ELISA shown in fig.
(2000) Electrophoresis 21. Invest. J.G. Specht. CrossDown Buffer is applicable for dif ferent immunoassays. 526-532  Miller. (2003) Clinical Chemistry 49. Genet.J. many interference effects can be minimized and innovative buffers for immunoassays make an essential contribution to it. J. Taken together. C. Lab. R. 14-17  Kusnezow. 10 ELISA with lysates of a human kidney-cell carcinoma. 1123-1144  MacBeath. (2002) Nat. that “good solutions” for these problems have been developed. (1992) J Clin Immunoassays 15. (2004) Clinical Laboratory International 28. J. 942-956  Span. 105-112  Kricka. The results shown here cover just a part of the different negative effects in different methods. G. Para Bioscience GmbH).Jr. Mol.F. 1708-1709 1 2 3 4 5 1 2 3 4 5 Abb. At today’s state of the technology. W.J. 32. respec tively. which is manufactured in reproducible qua lity with its wide spectrum of fragments of different molecular weights. Lane H: preimmun sera. © 2010 AppliChem • Immunoassay Buffer 9 . Left side PBS/NaCl/Tween® 20 and right side with novel CrossDown Buffer (AppliChem) Lane A-G serial dilution of immun sera.J.(7). Suppl. In addition. (Dr. nonspecific binding in immuno histochemical applications and falsepositive binding in immunoPCR can be prevented. Grebenchtchikov N. W. (2003) J.. Hoheisel. (1999) Clinical Chemistry 45. costs and time for optimizing assays can be avoided and reduced. Recognit. (1991) Scand.. During the last 30 years numerous molecular causes were found and the mechanism of interference investigated which led to the development of prevention strategies.. 16. can help to increase the efficiency of these methods. Geurts-Moespot. J. which could be minimized or even avoided with this novel buffer. Sweep. 165-176  Patton. 97-107  Wood.. It is new that the same sample and anti body dilution buffer allows to minimize different inter ference effects with different molecular principles at the same time.G. Clin. (2). Valdes. W.D.N. as well as reliability improved.J.. P. Column 5: blank. Literatur  Miller. J. L. 205.Conclusion The phenomenon of interference in immunoassays is as old as the application of antibodies for bioanalytical and diagnostic purposes. In fact one can say. The novel Blocking Solution I.(10).
Unfortunately. Hydrolysis of casein must be performed over many hours and any mishand ling may lead to precipitation of casein. falsify or even destroy results. Additionally applying AppliChem's CrossDown™ Buffer shall reduce or even abolish nega tive effects in different immunoassays such as immuno histochemical staining.blocking Without Blocking . Dr.. Nevertheless. It is based on highly purified. University Hospital Münster. All kinds of immunoassays require blocking to prevent nonspecific binding of antibodies or components of the sample to surfaces e. They all have in common that cer tain molecules are present in vast excess to cover the entire surface. A review of the literature shows that for every detection method using antibodies. Astrid Voigt. However. Dr. casein or synthetic molecules. if it contains molecules of different sizes where large and small gaps will be covered simultaneously. caused by matrix effects or cross reactivity. Blocking Buffer I . It is no won der that some antibodies will bind with high affinity to BSA. BSA blocking reagents 10 Immunoassay Buffer • AppliChem © 2010 . PARA Bioscience GmbH Many different blocking protocols exist and they work in various assays – sometimes better. Dr. Effi cient blocking means no areas on surfaces are available for nonspecific binding. Dr. ELISA plates or Western blot membranes. Rainer Klocke. Secondly..ready-to-use AppliChem now offers Blocking Buffer I which meets all criteria. AppliChem. the optimum reagent has to be determined for each new assay. Otherwise. Wolfram H. the blocking rea gent shall not react with or bind to any components of the sample or the antibodies.g. this binding would lead to a strong background. This ready-to-use buffered solution requires no assay optimization and is stabilized with ProClin® 300 instead of toxic additives like thimerosal or sodium azide. Tronhung Quang. Highly purified Caseinbased reagents have pro ven to meet most of the important criteria of the ideal blocker but the preparation of such a reagent requires experience of a skilled person. Dr. even effective blocking may not pre vent some background. Marx. the devil is in the details. Prof. Ideal Blocking What properties are a must for the ideal blocking reagent? First. In this case. gelatin from fish. since they all have certain restrictions when used with real samples such as blood. No Result! This is a short message describing the necessity to block surfaces in immunoassays. Sigrid Nikol. blocking would lead to an even increased background due to the reaction of antibodies with the blocking reagent. cell lysates or tissue sections. In practice. nonfat dried milk. That's the theory. pose special problems since many analytes are coupled to BSA to achieve a better immunization. universal blocking solutions do not exist. sometimes worse. serum. Dr. it has to completely cover the surface and this is achieved best. University Hospital Jena. Western blots or ELISAs. several hundred blocking protocols exist describing variations of blocking solutions based on different blocking reagents. Christoph Specht. Frequently used blocking reagents contain BSA. chemically modified casein with an optimum distribution in terms of fragment size.
Germany) detects immunoglobulins from guinea pig and is used for immunotoxicological studies with guinea pigs. columns 7–12 10 ng/ml). heart brain Western-Blot Detection of the antigen CaMK II in lysates of heart and brain tissue of C57Bl/6 mice by Western blotting.31–10 µg/ml in PBS).Just theory? This is the practice! heart brain Immunohistochemistry The antigen Nestin was detected by ABC-immunocytochemical staining with alkaline phosphatase on Cytospin preps of the neuroblastoma cell line SK-N-LO. Gronau. PBS-BSA buffer was used as a blocking buffer and detection was c arried out with streptavidin-peroxidase (Sigma) and ortho-phenylenediamin (Sigma). p roteins were transferred to a nitrocellulose Optitran BA-S 83 membrane (Schleicher & Schuell) and detected by ECL. After separation by SDS-PAGE. While a standard blocking buffer based on 1 % BSA in PBS stained large parts of the whole surface (upper panel). and the secondary antibody a polyclonal HRP-conjugated antibody (Santa Cruz). Correct staining of the cytoplasmic Nestin is now clearly separated from the hematoxylin staining of the nucleus. with Standard Assay Buffer with CrossDown false-positive binding false-positive binding ELISA This ELISA (developed by PARA Bioscience. Inc. made the identification of the bands possible. (concentration range each 0. © 2010 AppliChem • Immunoassay Buffer 11 . Use of CrossDown™ Buffer abolishes false-positive binding and allows a concentration-dependent detection (lanes B to G1–6 and B to G7–12). Blocking with 5 % non-fat dried milk in TBS-Tween® is shown on the left side. The capture antibody Goat-anti-guinea pig-IgG F(ab')2 and detection antibody Goat-anti-guinea pig-IgG F(cγ) biotinylated were from Jackson ImmunoResearch Laboratories. A combination of B locking Buffer I and CrossDown™ Buffer as antibody dilution buffer for the primary and secondray antibody (right side). The primary antibody was a monoclonal anti-CaMK II antibody (BD Bioscience). Guinea pig IgG was diluted either in CrossDown™ Buffer or PBS (columns 1–6 50 ng/ml. using the ready-to-use Blocking Buffer I led to a reduction of background staining (lower panel). The false-positive binding observed in the c ontrol lane A1–A12 and the blind values (lane H1–H12) prevents i nterpretation of the assay. This Western blot cannot be interpreted.
prepare small aliquots and freeze. in biomedical laboratories sodium azide (final concentration 0. because nearly every antibody cannot be frozen without losing part of its activity. The antibody is always ready-to-use for your next experiment as you don’t have to slowly thaw. Don’t re-freeze and thaw again. Selection Guide CrossDown Buffer Sample Buffer T+ Sample Buffer TBlocking Buffer I Blocking Buffer II EGrade Blocking Buffer III BSA ELISA. You may dilute the antibody in Antibody Stabilizer and directly store at 2-8°C.02-0.005 %) are added to reagents. EIA. RIA Western Blot Protein Array Immunohistochemistry Immuno-PCR 12 Immunoassay Buffer • AppliChem © 2008 Washing Buffer TrisT+ Washing Buffer TrisTCoating Buffer Stripping Buffer I 12 Immunoassay Buffer • AppliChem © 2010 . In case the test is positive. as the loss of acitivity will be greater with every freeze and thaw cycle. One of the main advantages is that you don’t have to aliquot for storage. because albumin stabilizes antibodies. we recommend the use of our Antibody Stabilizer (prod. ProClin® 300 may serve as a substitute. Is it possible to store antibodies in 50 % glycerol? Does it make sense to add albumin? Principally. as thawing too fast may damage your antibody in solution. you simply may take just the quantity of antibody you really need right now.2 %) or Thimerosal (final concentration 0. A modern way of long-term storage of antibodies is storage in our reagents Antibody Stabilizer-PBS or -Tris. Be careful during thawing. Most antibodies show extremely long shelf lives. it is good to add albumin. If you really want to freeze antibodies. if growth of bacteria and fungi/molds has to be prevented. For any new assay. A7148 and A7135). we recommend testing the performance of the antibody after thawing. because they contain special ingredients stabilizing the structure. if stored in Antibody Stabilizer of around 5 years or more. Both substances are toxic and less harmful alternatives are wanted. no.tips & tricks Which substances may be added to antibody-containing solutions as preservatives? Frequently. For long-term storage of antibodies in solution. For such applications.
1. Comparing DNA-ExitusPlus™ to conventional products. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Likewise. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. They are available in anionic (A). despite their corrosive or even toxic properties. all DNA fragments will be destroyed. completely different story. Eight different basic structures with precise absorption and emission maxima were selected to cover the full working range of wavelengths. bacterial colony will be white. but when ELISA are validated (e.5 g water). a further. a pair of fragments differing by 4 bp is usually resolved on less than 4 cm of gel length. (iii) size of the DNA inserts. They are also applied in the screening of cytotoxic agents and lymphocyte activation. Primarily. for denaturing. relatively small dyes with good absorption coefficients in the difluorides (BDPs) are chemically and pH insensitive. Stir solutions CO gently for precise measurements of the pH value. AppliChem now offers a product for use in every research in volumes starting as small as 50 ml. 3 mm each. Dipyrrometheneboron robust. fast and highly efficient positive cloning system of blunt or blunted PCR fragments. cationic. which describes the change of the pK at an increase of temperature by 1°C. E. No. reduction of contaminant concentrations constant HPLC-retention time save solvents DNA in living cells. regardless of their size. The separation of three PAHs tests were performed over a period (Polycyclic Aromatic Hydrocarbons) with solvents. urea-containing SeparateIT Polymer Solution comes as a 10X solution. pH insensitive dyes with high and photochemically absorption coefficients and fluorescence quantum yields. that also employ positive selection of recombinants poly-linker (MCS) in the middle of the include: (i) The position of the toxic gene. including the multiple As a result of this breakthrough.0 g water. This uncontrolled evaporation of solvents is not only a potential health hazard but also the loss of solvent by evaporation interferes with the quality and reproducibility of HPLC runs. Refolding antibody conformation of some of the of coated antibodies and 2. down to the Detection limits are lowered further with CheLuminate-HRP FemtoDetect. We recommend that Mini gel cassettes. (ii) Under stirring dissolves in the solution. A buffer keeps the pH of a solution constant by taking up protons that are released during reactions. (PDA). DTT.2 kb. the commonly used amine reactive and thiol reactive groups) while retaining the photophysical features of the parent dye. A grade “25” means that the dry matrix will take up 2. the difference can be measured in many assays. The size exclusion cut-off for AppliXchange-G25 M is set at 10 kD for proteins and 10 bp for nucleic acids. which in turn retains only the smallest molecules. AppliChem partnered up with this multiBIND Cologne. which lab is called the AppliCoat Plate Stabiliser (Cat. 125 x 4 mm HPLC-Column Purospher AppliChem provides background information or published application notes for some selected products. gel staining and recording are carried out as usual. Bottle D This bottle was closed with a cap having 3 holes. a corrosive and toxic substance. this could help to decrease such variabilities in assay performance. Thanks to this increased spacing. which are 8 or 10 cm long. EDTA. To address issue. The incorporation of a No. The chemical interaction of gel matrix and molecules be separated is negligible. or by releasing protons when they are consumed by reactions. Yazynin et al. multi-step reaction. the MCS of a positive selection vector Ligation of a PCR fragment into disrupts the expression/activity of the toxic gene. HRP catalyzed oxidation of luminol is a complex. This is due to longer path the smaller molecules must the travel. one can often already smell the solvent vapors. without.2]. which really differ from batch to batch or even can from well to well. 1). 2001) or according to other validation strategies. the techniques is suited to concentrate samples. The and therefore. acenaphthopyrrole and cyanine top-notch brightness perfor- nce (MCS) of pBARN vectors. resolving power of SeparateIT gels is at least twice higher compared to resolving power of any other gels. Get your free copy! Orders by fax 06151/93 57 11 by eMail service@applichem. has since been honed and the late simplified. The pBARN PCR Blunt Cloning System is based on the toxic barnase gene product. Such relatively short gels will be previously required 20 –30 cm long polyacrylamide appropriate for the majority of demanding separations which have gels. fitting precisely the standard GL45 glass bottle thread used. It is therefore usually necessary to stabilise the H concentration in vitro by adding a suitable buffer to the medium. Therefore. 2. false-positive clones are frequently detected. AppliCations Colorada: A New Generat Of Fluorescent Labels ion The Colorada product line today. commenced in the 1950s. its pH may vary with temperature. The use of tetrazolium salts. we investigated the molecular mechanism of action of various commercially available DNA decontamination reagents. This is a key benefit of AppliCoat Plate Stabiliser. This stabiliser solution is easyto-use and has a great advantage compared to almost any stabiliser used in industry. and lower consumption gel staining reagents. Only in living cells mitochondria are capable to reduce XTT to form an orange colored water soluble dye.4 kb and 3. Keywords XTT assay cytotoxicity testing non radioactive assay quantitating and viability testing of cells The need for a reliable. And there is a second benefit with this product: Two benefits with one solution When antibodies are coated onto ELISA plates. the main component of these systems. These benefits are used for production of high-quality ELISA kits as well as in research applications Stabiliser the percentage of active antibodies now. (iv) High-concen azide. a new level of chemistry has been developed to provide the same fluorescent label with different charge (anionic.g. Likewise. improving bioanalytical assays to the highest level. With home-made ELISA it is a For any new measurement one has to coat a new plate. water-soluble polymers. such as incorporation led to the of radioactively labeled H-thymidine into DNA or the use of 5-Bromo-2‘deoxyuridine (BrdU) as a substitute for radioactive thymidine to label Enhanced ChemiLuminescenc e Detection Kits for Horseradish Peroxidase in Western / Southern / Northern Blotting The peroxidase-catalyzed nm. Fig. Bottle B This bottle was tightly closed with the GL45-cap provided including a Teflon-foil seal. provide femtogram (10 )-level Long Signal Duration – All substrates exhibit long light emission.2]. In addition. E. Remaining fragments are fully destroyed after 10 minutes. It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. a beaded composite material It exhibits high selectivity. but do not destroy the genetic information encoded in DNA strands and there is the risk that they may be chemically re-activated. cationic (C) and zwitterionic (Z) form. The larger molecules can not enter the pores and therefore have a shorter path. The number “25” correlates to the “water regain” of the matrix. The polymer is not optimized polyacrylamide gels.7 Keywords Keywords Immunoassays Antibody Stabilisation ELISA Plates Gel filtration matrix Why is there such a great difference in storage between home-made ELISA and ELISA kits? The reason is that in professional ELISA kit production the plates are not only blocked after coating. No absorption to takes place. But there were no such high quality stabiliser lutions freely available in low volumes for souse in research lab until now. Many biochemical processes are markedly impaired by even small changes in the concentrations of free H ions. because after storage of some stored for two years at 4°C without any problem. The water regain/extent of cross-linking affects the separation properties of the Larger water regain/less cross-linking gels. To meet the need of labeling the broadest range of target molecules each fluorescent compound is available with many different reactive groups (e. including Applichem’s SafetyFirstCaps for HPLC. chromatograms of a mixture of the three PAHs (Naphthalene.e. Therefore. because unique method of action is based on chemical its and not enzymatic activity. (Acenaphthopyrrole dyes) are chemically robust. Buffer and pH effects on resolution are minimal. We found that when using high concentrations of DNA and short incubation times. after several minutes. which in turn retains larger molecules. The positive effects of AppliCoat Plate Stabiliser are shown in Fig. was compared. b-Mercapto to ethanol) changes in the pH should also be considered and pH should be retested. all bottles were kept at room temperature under a fume hood with a gentle air flow for 31 days. leaving not a single class of fragments. Such effects depend on the used antibodies. These dyes have high absorption coefficients in the far red and good quantum yields of fluorescence the near infrared (NIR) spectral region. The nicks will be introduced statistically at any site. equipment and the environment. results in larger pores. How can microbial contamination of buffer solutions be prevented? (i) Sterilization by filtration through a 0. to a few restriction sites for vectors combine advantages oxidation of luminol produces so-called enhancer into the Keywords high absorption coefficients high fluorescence quantum yields wavelengths from 400–850 nm coumarin BDP acenaphthopyrrole cyanine Coumarin dyes are chemically and photochemically near UV / blue spectral region. Slow addition of a strong acid or base whilst stirring vigorously avoids local high concentrations of H or OH ions. The complete decontamination of equipment and surfaces from any DNA molecules is important for biological containment and safety. This polymer has been optimized for the gels with a acrylamide to N. and coefficients and excellent fluorescence quantum yields. a grade of “50” gives a water regain of 5 grams (1 g AppliXchange-50 plus 5. Less cross-linking gives a more is flexible bead and allows additional swelling. 1999. respectively). 670 LSS and Colorada 690 LSS) belong to the chemical class of heptamethine indocyanines as well. (ii) Addition of 0.5 times its weight of water (1 g AppliXchange-G25 M absorbs 2. (iv) In some cases Limited handling is cumbersome. Bottle Keywords Molecular Cloning Vector PCR Blunt Cloning Positive Selection Barnase Introduction Many cloning vectors in use today employ a blue/white screening approach to identify successful fragment insertions. The above methods have a number of disadvantages. Keywords improved resolution Polyacrylamide Gel Electrophoresis SeparateIT Polymer Solution The extraordinary resolving power of SeparateIT gels results from addition of a special polymer to a polymerizing containing a monomer and a cross-linker.COOX. azide (. mobile phase. complex such as MTT. conformational changes can occur due to surface-protein interactions. It is just as simple as a “second blocking step”. it is applied to the separation of biopolymers such as proteins and nucleic acids. Gel polymers are arranged in a conceptually different way. i.Give it to me! AppliCations No. the contrast to other positive cells that contain non-recombinant AppliCations No. Less water regain/more cross-linking results in smaller pores. AppliCations Improving Separation During Electrophoresis SeparateIT gels represent a novel gel matrix for DNA No. cytokines or media components. Gels with SeparateIT Polymer Solution are prepared in the same way as any regular polyacrylamide gels. The extent of cross-linking has been optimized for a particular water regain. developed in seventies. Most decontamination reagents are based on several molecular principles for the destruction or inactivation of material: Modification and denaturation genetic can mask. dyes good quantum yields of fluorescence and are available in anionic (A). Not to be mixed up with gel electrophoresis. AppliCations Biological Buffers AppliCations No. Pentamethine cyanine have extremely high absorption coefficients. and Autoclave-ExitusPlus™. with specific formulations to meet the various requirements for immunoblotting optimal substrate for your application). The increased resolving power of SeparateIT gels enables full separation of closely spaced bands on short gels. The pBARN PCR Blunt Cloning System is an easy-to-use. 6 kb plasmid) only a small fraction of fragments with 200 to 500 bp in size will remain after 5 minutes.12 Cell Proliferation Assay XTT Cell proliferation assays are widely used in cell biology for the study of growth factors. It is therefore advisable. (ii) Limitations for the bacterial strains to be used. This handout summarizes the most commonly used buffer substances and respective physical and chemical properties. to mid-femtogramlow picogram (10 ) range. faster electrophoresis runs. The principle of positive selection cloning is rather straightforward. SeparateIT gels selectively retard the migration of large molecules. including: use of radioactive materials and relatively techniques. while the ultimate FemtoDetect Plus. Dye structures were is a selection of the newest carefully chosen to provide No. (see table below for selecting the Outstanding Sensitivity – The intense light output generated by the CheLuminate-HRP corresponding improvement in sensitivity.coli upon plating. by addition recent of a suitable catalyst. This traditional method. once expressed. a small. Assuming a theoretical smaller nicking activity of 100. the separation of the ones is very effective and is performed under mild conditions. There is a relationship between the delay this causes and the molecular size.5. Thus. Preserving correct conformation during storage. if not. its effects on fragmentation are totally independent of the size and sequence of the DNA fragments.g. is based on the fact that living cells reduce tetrazolium salts into colored formazan compounds. products that relied on modiSequence-Independent Degradation of DNA Only Applichem’s DNA-ExitusPlus™ is capable of achieving rapid and efficient degradation of nucleic acids. The Matrix AppliChem‘s size-exclusion chromatography columns are packed with AppliXchange-G25 composed partially of polymerized dextran.COOH). large increase in light output is obtained: In analytical laboratories. whereas only partially degraded DNA pieces. This is the case because DNA bands that migrate a short distance sharper than the bands that migrate remain a long distance. because the antibodies can be affected. in accordance with a new theoretical model of gel electrophoresis. however. transformed competent a plasmid E. were com with the reference. N-terminally fused to an especially designed sequence. days the plates don’t perform as well as before. performing analytical restriction enzyme digest or sequencing. On entering an HPLC laboratory. For instance the physiological pH value for most mammalian cells at 37°C is between 7.02 % (3 mM) sodium (iii) Storage at +4°C. as far as possible. M. which is designed to detection. HPLC-System HITACHI LaChrom Elite system with Diode Array Detector under control of the EZChrom Elite Isocratic pump conditions with premixed Software. The smaller molecules enter the pores and go inside the beads. Therefore. Heptamethine indocyanine dyes have extremely high absorption coefficients and good quantum yields of fluorescence in the near infrared (NIR) spectral region. • Using Bottle B as a reference. Trimethine cyanine dyes have very high absorption coefficients. zwitterionic and neutral). into a glow and greatly improves the analytical characteristics of the reaction in terms of increased signal intensity and duration [1. (v) If other components are added the buffer (e. cationic 8-Oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles (c) and zwitterionic (Z) form. While enhancers are useful in improving enzyme turnover and increasing the equilibrium concentration of a key intermediate. DNA fragments (such as PCR products or pieces of a genome) are inserted into a multiple cloning site (MCS) within DNA vector. including lower cost of the gel materials.1 gy gene technology demonstrate Nucleic Acid Decontamination with The ExitusPlus™ Technolo Advanced experiments in that even small amounts of free DNA molecules are sufficient to cause infections. allow direct selection of recombinants via disruption of the lethal barnase gene. pBARN cloning are not restricted to the use the ligation of inserts only. NHS ester). a lower of amount of sample DNA needs to be loaded on a gel with a high resolving power compared to a gel with a low resolving power. and cationic The dyes with a Large Stokes Shift (Colorada (C). Test Conditions A This Bottle was closed with a SafetyFirst Cap.2 times however. The vector contains the barnase gene under the control of the LacZ promoter. starting from 400 nm up to 850 nm. This results in limitations regarding the choice of restriction enzymes well as limitations of using ‘universal as sequencing primers’.. the family of AppliChem‘s CheLuminate-HRP chemiluminescent substrates has been developed. active ester triazine. high resolution purified with AppliXchange-G25 M are and chemical stability. according to “Guidance for Industry: Bioanalytical Method Validation”. including polyacrylamide gels. The results of the tests were dramatic. aliphatic amine (. stored in either tightly bottles (SafetyFirst Caps) or bottles closed closed with caps with holes of different diameters. 1996. and are available in anionic (A). Chrysene).6 Improving quality of ELISA Using ready-to-use ELISA Size-Exclusion Chromatograph y for purification of biomole cules No. (iv) Setting of the ionic strength of a buffer solution (if necessary) should be done in the same way as the setting of the pH when selecting the electrolyte. They have high absorption photochemically robust. (vi) In the presence of divalent metal ions carbonate or phosphate buffers may form precipitates . In order to satisfy numerous requests from researchers who wish to improve resolving power of their acrylamide based AppliChem now provides SeparateIT gels. the buffer substances may undergo chemical changes that inactivate them or modify them so that they have an inhibitory action (Ellis & Morrison 1982).). In order to determine the number of viable cells Cell Proliferation Kit XTT employs 2. Fragments generated by either Taq DNA polymerases or other DNA polymerases without proof-reading activity require a filling-in reaction for optimal cloning efficiency. it still has two major drawbacks: low efficiency of blue/white selection and high costs per cloning reaction. affecting the functioning of the system under investigation. the pH value is set using NaOH/KOH or HCl. the latter being very useful Furthermore. Keywords Nucleic acid decontamination DNA degradation test Autoclaving DNA PCR test All or Nothing at All Destruction and elimination of nucleic acids depends to this day. First. of 31 days. since this value increases depending on the electrolyte used. We present here quantitative data comparing the efficiency of various sealing methods. It should be mixed with a buffered solution of acrylamide and prior to addition of TEMED and ammonium Bis persulfate. making necessary sub-cloning and further time consuming investigation of candidate colonies. The separation efficiency is electrophoresis.) The ability to absorb water is dependent on extent of cross-linking. as well as preventing artifacts in PCR amplification experiments. most of the antibodies are not active. Molecules separated according to size. A7708). Procedure • At the start of the test. typically with beads of dextran or agarose serving as gel matrix. we can demonstrate that it is fast and efficient in destroying nucleic acids without harmful or toxic effects on lab workers.g. dipyrromethene borondifluoride. Temperature Practical Tips – Preparing Buffer Cross-reactivity of the pH value of a buffer and Solutions Interfering effects storage conditions Depending on the buffer substance. cloning systems BARN-1/-2 of special E. Connecting tubes are inserted into the bottles through the holes in the foil/cap without any additional sealing. These differences from well to well can affect the variability of an assay. pared • After the first measurement. so that DNA bands remain sharp but are more spread out relative to each other. easy to perform process has been an industry This standard for thirty years. However such basic covers do not seal the solvent containers efficiently.10 blue/white screening is not required for this system. the most popular being p-iodophenol and p-hydroxycou- a weak flash of light at 425 buffer forces the flash signal without the known disadvantages of other cloning systems Keywords Luminol Redox Mediator Enhancer Long light emission The enhanced. Only around 2–8 % of all coated antibodies active and can bind to analytes and this remain is greatly variable depending on the surface characteristics of the ELISA plate.g.9 mances. RP-18e (5µ). If the insertion was successful. does mean biomolecules large molecules from the small volume of the eluate may be kept small keep their biological activity. However. none of the conventional reagents destroyed DNA molecules efficiently. Size-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. The temperature dependence of a buffer system is expressed as d(pK )/dT. It gives better storage stability for coated antibodies and antigens than most other products do. (if known) Address Title | First Name | Name ZIP Code City | State Department Country Institute Phone Fax University | Company eMail © 2010 AppliChem • Immunoassay Buffer 13 . were found in the decontamination fication or denaturation methodology.N-methylene-bisacrylamid ratio of e of 29 : 1. To facilitate more simple and efficient high-throughput cloning several positive selection cloning systems have been developed (Table 1.coli cells are grown in the presence of X-gal. the electrophoresis.8 AppliCations CheLuminate: Improved Chemiluminesce AppliCations AppliCations SafetyFirst Caps: Safety-Systems for HPLC pBARN Cloning Vectors – Positive Selection of Recomb inants The BARN PCR Blunt Cloning barnase gene expression Systems -1 and -2 are based by cloning of PCR fragments on the disruption of toxic into the multiple cloning site vector are killed In No. This system is also called gel filtration.de Customer No. This is a water regain of 2.22 µm filter unit or by autoclaving. comprising DNA-ExitusPlus™.4 kits from manufacturers is easy and convenient. the pH value can also be set by mixing the two substances. are used for casting acrylamide-Bis gels with SeparateIT Polymer Solution. recombination or biological transformation [1.N ). to develop a new and unique nucleic acid decontamination technology: the ExitusPlus™ family of products. iodoacetamide and 2-pyridyldisulfideamine (. The reactive groups include carboxylic acid (. this feature has been specifically maximized in the formulation developed for the CheLuminate-HRP FemtoDetect substrate. values It is thus easy to find the ideal product among the Colorada series starting from the desired absorption/emission peak maximum and subsequently decide both the reactivity and charge properties. the concentration of the dye is proportional to the number of metabolically active cells.11 fluorescent labels available No. tration stock solutions. Somethere is no kit available with kits such as their limits of good suppliers may be ELISA is required because characteristics of the available Ready-to-use ELISA kits from AppliCations No. Then. Polymer Solution. to set the pH at the working temperature to be used for the investigation. it is seen that an efficient and total fragmentation was only obtained with DNA-ExitusPlus™. it is common practice to „seal“ bottles of HPLC solvents simply with a perforated aluminium foil or plastic cap.3-Bis-(2-methoxy -4-nitro5-sulfophenyl)-2H-tetrazolium-5-carb oxanilide salt (XTT). safe and complete DNA decontamination depends on the degradation of DNA into very small fragments. Advantages of SEC/Gel Filtration Some important features of the technique have to be mentioned. of positive cloning systems available.e. FDA. A sandwich ELISA with a monoclonal antibody has been cross-linked dextran beads Desalting Buffer exchange Nucleic acid/ protein purification Keywords chemical properties usefull pH range buffer preparation Recommendations for the setting 1. Two DNA fragments in the 70 –150 bp range that differ by 1 bp can be separated on SeparateIT gels that are 8 cm long. leaving an opening of an area of approximately 0. If there way of refolding antibodies and of preserving was a antibodies from conformational changes during storage. for several reasons. chemical composition could be prepared with several and molecular weight of the polymer required a careful optimization for each particular monomer/cross-linker combination.coli cells that contain non-recombinant Disadvantages of known cloning systems vectors are killed upon plating. For example.0 and 7. Applying this theory to a test molecule (ccc form.coli strains nor In summary. 3. Tighter cross-linking the limits the ability of the beads to swell and as a consequence the water regain lower. the luminol radical anion. Using new methods that detect extremely low levels of DNA molecules. Even with AppliCoat Plate will still be in the range of 2–8 %. i. on bleach.NH ). Pyrene. Titration (i) Generally. Figures 1 and 2 show the results of comparing produced by the novel DNA-ExitusPlus™ the fragmentation process with conventional reagents. It is always advantageous when closely spaced bands separate after migrating just a few centimetres. which offers a 24-hour light emission least ten times longer than with standard – at phenolic enhancer substrates. Bottle C This bottle was closed with a cap having a 10-mm hole leaving an opening of an area of approximately 0. Using highly sensitive detection assays. primers). If this is not done. Smaller fragments will disappear before the larger ones. level in sensitivity. some of which contained complete genetic information. The use of shorter gels is beneficial easier gel preparation and handling. Keywords health protection To test for the reproducibility of the retention time and the loss of solvent by evaporation. Thus it has two benefits: 1. However. The longer path arises from the pores of the beads.5. can Entering the pores creates a longer path for the smaller molecules. sensitive and quantitative assay that would enable analysis of a large number of samples development of methods. substrates translates into a Even the economical CheLuminate-HRP PicoDetect Extended formulation provides a sensitivity level at least eight times higher than with phenolic enhancers. Larger plasmids require a longer incubation time than ones (e. Smaller molecules pass significantly slower through the column than larger molecules. For stabilisation of a plate one has to incubate with a coating stabiliser solution. pBARN-1 and -2 PCR Blunt Cloning Vectors (3. (iii) If a buffer is available protonised form (acid) and the non-protonised in the form (base). When the antibodies (or any proteins) into close contact to the plastics surface come of the ELISA plate. permitting growth of positive binants (up to 100 %) upon transformation recomonly. maric acid. Typical enhancer compounds are substituted phenols. In addition. solution While the gels with SeparateIT-like properties different polymers. Mg . work  has shown that. Purified biomolecules significantly diluted when processed using are not AppliXchange-G25 M. but also stabilised. is achieved with CheLuminate-HRP (10 ). all 4 bottles were filled with the same mixture of Water + Methanol = 20 + 80 (w/w). dichlorofor labeling -SH in peptides or proteins.000 nicks per minute. The result is that most antibodies coated on a plate are unfolded or inactive. home-made the right antibodies or the detection are not appropriate. highly active ribonuclease from Bacillus amyloliquefaciens (Yazynin et al. the the colony will appear in blue color. The larger molecules therefore travel faster through the column than smaller molecules. in with a large stoke shift (> 100 nm). The Colorada structures are based on 4 different chemical classes: coumarin. SEC does not require electric current and the sieving effect will not separate small molecules first. there are big differences in terms of the separation principle.212 cm .785 cm .
which seemed to work well by inclu ding a BSAbased blocking reagent (Blocking Buffer III BSA). or the results have to be reliable. phosphatase or fluorescent dyes. Wolfram H. detection antibodies or the label such as peroxidase. but only when they are prepared using casein which has undergone severalfold cleavage. Some do interact with e. ELISAs. expensive. Looking at the CV as shown in fig. efficiency is comparable. The only option avai lable is based on ultrapure BSA and in many test systems.e. is a reliable result achie ved. At first view. several very different methods for blocking were developed and became established especially proteinbased blockers containing casein or BSA.e.g. the saturation of free binding sites on surfaces of ELISA wells or Western Blot membranes. i. Most of these substances do not influence the results. Not until running the same assay using AppliChem’s Blocking Buffer I. combining quality and economy. Applichem's Blocking Buffer I). Western Blots or other immunoassays simply serve as tools to verify or falsify a hypothesis of the whole research project. Upon first glance. As soon as inter actions occur. Figure 1 shows a sandwich ELISA. The relatively large BSA leaves gaps on the sup port surface which will be covered by smaller molecu les coming from the sample. In most cases. Noncomplete blocking will be cursed with high background and either useless or nonmeaningful results. AppliChem Blocking in immunoassays is an important step. no serious alternative to casein exists. The diffi culty is that one cannot predict when it will happen. Whenever reproducible results with a low standard deviation are of importance. These differences are mainly due to differences in molecular size of the blocking agent. ultimately influences the significance of all subsequent tests.blocking Comparing Blocking Reagents Quality and economy now combined in a novel blocker Dr. how strong the interference will be and whether devia tions will shift results up or down. But CV’s tell something different. Marx. Differences appear in terms of standard deviations when real samples are measured (determined as coefficient of variation (CV) for multiple measurements). they lead to erroneous results. preparation is complex making this product more expensive. 1. The caseinbased blockers show an unsurpassing efficiency. as shown for the BSAblocked 14 Immunoassay Buffer • AppliChem © 2010 . what appears as an unimposing effect. high CVs. the problem is obvious. then there exists no alternative to the highly purified caseinbased blocking reagents (e. You can identify such interferences simply by observing high standard deviations. State of the art If samples are rare. Blocking Buffer II EGrade.g. the assay looks good but the results are unre liable. Unfortunately. Now. Therefore. i. Those who use unreliable tools. we present a new alternative.
one problem couldn't be solved: reproducibility of results thereby limiting their use. doubts are justified. Ne vertheless. Due to the uncontrollable quality of these raw materials. synthetic blockers were investigated and Tween® represents the first. is reliable and simple. The pro blem is that a large number of commercially available antibodies as well as antibodies generated by and exchanged between scientists not only bind the hapten with significant affinity but also BSA. Other synthetic blockers are also availavle at low prices as well. 15 . Blocking Buffer II EGrade is manufactured to DIN ISO 9001:2000 quality requirements. In some laboratories and assays they meet the requirements and. To determine the CV a complete ELISA plate was measured (n=96). preclinical or clinical studies or in diagnostics. There is another problem arising from time to time that must not be overlooked. while quality is acceptable – in contrast to synthetic blocker. The ELISA was performed once with Blocking Buffer I and once with Blocking Buffer III BSA under identical conditions. in the present case. it is obvious why very few scientists would trade quality for marginal savings. The big disadvantages of good proteinbased blockers is the high costs and the search for alternatives has resulted in several varieties. Gelatin from fish or nonfat dried milk are just two to mention. best known and simplest solution. in most cases BSA is a very good blocking reagent. The costs are comparably lower than for proteinbased blockers. While consistency of raw materials may be optimal. Since the synthesis of the peptides is controlled. Unfortunately. to BSA as carrier molecules to generate anti bodies. repro ducible quality but at low cost. since reproducibility is not given. the exact opposite is reality and research antibodies do not include this essential information.ELISA. © 2010 AppliChem • Immunoassay Buffer coefficient of variation [%] Blocking Buffer I Blocking Buffer III BSA Fig. Over the years it has been common practice to couple small antigenes. free of any serum pro teins. It works well. must be prepared that their results will be ques tionable and. socalled haptens. AppliChem presents THE new alternative for efficient and costeffective blocking in immunoassays. In those cases. blocking is uniform and repro ducible from batch to batch. in many other assays they constantly fail. and peptidebased.g. This wouldn't be a problem if the producers of such antibodies would mention the method of immunization. Repro ducibility of an assay depends on constant quality of the raw materials. in pharma assays. It is cheaper than optimized caseinbased blocker. Alternatives wanted Since the desired alternative needs to be of high. Now. 1 Comparison of the coefficient of variation (CV) of an ELISA with blood samples. e. these blocking reagents are not used in medical research. even if the “BSA" purification is laborious. one would be aware of the potential interference and would not choose a BSAbased blocking reagent. Considering that the total costs of blocking are just a small part of the total costs of an assay.
2a. The left column is the control without blocking (K-). The order of blocking reagents is as in fig. CV [%] Blocking Buffer I Blocking Buffer III BSA Blocking Buffer II Egrade synthetic Blocker Fig. This is a modified analyte-free assay originally developed by Steinitz & Baraz (2000). according to FDA guidelines). the more reliable the assay. 2b Image of the ELISA plate described in fig. 2a OD-values for blocking over night with different blocking reagents. K- BB I BB III BB II SyB Fig. 2a Blocking Buffer I (BB I). 3 Graph of the coefficient of variation (CV) of the assay from fig. 2. In industrial practice.OD 450 [nm] Blocking Buffer I Blocking Buffer III BSA Blocking Buffer II Egrade synthetic Blocker Fig.g. The lower the CV. Blocking Buffer III BSA (BB III). Please note that the CVs shown here only represent effects of blocking reagents. 16 Immunoassay Buffer • AppliChem © 2010 . since the assay don't includes analytes and other negative effects. CVs of < 15 % for ELISAs with real samples are needed (e. measuring the influence of blocking without negative effects of real samples. Blocking Buffer II EGrade (BB II) and synthetic blocker (SyB).
where Blocking Buffer II EGrade gives a low background. milk powder or synthetic blocking reagents. It is question able whether a reproducible assay may be established with such a synthetic blocking reagent. Methods 238. & Baraz. Blocking is highly reproducible with a constant CV. 2b the correspon ding image of the blocked ELISA plate.  Steinitz. highly purified and multiply digested casein. After washing. The comparison according to Steinitz also proves the unique position of proteinbased blockers for sensitive applications like pharma assays. Quantitation of the Blocking Effect of Tween® 20 and Bovine Serum Albumin in ELISA Mircrowells. All variants do block. A rapid method for estimating the binding of ligands to ELISA microwells. Blocking Buffer II EGrade combines economy and reproducibility for various assays. The advantage of the novel Blocking Buffer II EGrade is clear. Such a CV is transferred to the total CV of an assay according to the error propa gation of each separate error of the assay. artificial test system without any interfering substances was used. The blocking efficiency of the proteinbased blocking reagent is superior. but using peroxidase) without any additional additives. The left column served as an unblocked control. synthetic blocking reagent. (2000) J. the wheat separates from the chaff. Here. Biochem. Literatur  Steinitz. 282. as well as a commercial. reproducabillity is seen also. Conclusion For many assays in LifeSciences. The coefficient of variation of the same experiment is presented in fig. M. L. The graph in fig. The lowerthe optical density (OD). Blocking Buffer III BSA based on BSA. the novel Blocking Buffer II EGrade. Blocking Buffer II EGrade has the potential to combine economy and quality. the synthetic solution looks pro mising. eight determinations instead of double values. and may be regarded as a suitable alternative to the proteinbased blocking reagents. Combining the results shown in fig. identical to the proteinbased reagents. the CV value of 25 % is extremely high. fig. the better the blocking efficiency. © 2010 AppliChem • Immunoassay Buffer 17 . The surface of the ELISA wells were blocked by the different blocking reagents and incubated with a detec tion antibody (analog to Steinitz (2000). a test system was established with the following changes: peroxidase was used instead of phosphatase as reporter enzyme. At first view. clinical studies or other assays inclu ding valuable samples. (2000) Anal.Analyte-independent comparison Is it possible to determine the real blocking efficiency and quality of a new blocking reagent? According to the methods of Steinitz & Baraz (2000) and Steinitz (2000). 3. 2 and 3 allows one to draw the conclusion that in those assays. This is an advantage over other alternatives like the fish gelatin. Immunol. good for Blocking Buffer II EGrade and also good for the synthetic product. 143-150. and additional determination of the coefficient of variation (CV). AppliChem offers an alternative to high cost proteinbased blocking reagents. 232-238. Figure 2 shows blocking overnight. The test included Blocking Buffer I. M. Even though a simple. 2a shows the ODs. the staining reaction was carried out.
But there were no such high quality stabiliser solutions freely available in low volumes for use in research lab until now.g. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2-8 %. conformational changes can occur due to surface-protein interactions. An improved workflow for most labs This advantage of a prolonged storage time can help to set up a completely new workflow in research labs. With homemade ELISA kits it is a completely different story. This monoclonal AB lost its binding activity when coated on a plate and stabilised only with BSA without applying AppliCoat Plate Stabiliser. Whilst nowadays it is best practise to produce a new and fresh ELISA plate whenever some measurements with a homemade ELISA are done. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate. according to “Guidance for Industry: Bioanalytical Method Validation”. Blocking Buffer I. For any new measurement one has to coat a new plate. this could help to decrease such variabilities in assay performance. These differences from well to well can affect the variability of an assay. Refolding of antibody conformation of some of the Industrial technology now available even for small research labs According to the needs of research labs AppliCoat Plate Stabiliser (A7708) is available in volumes of 50 ml. Thus it has two benefits: 1. Test it for yourself! 18 Immunoassay Buffer • AppliChem © 2010 . because the antibodies can be affected. coated antibodies and 2. FDA. Those plates are dried in an incubator at 20°C or 30°C for 60 to 120 minutes. the difference can be measured in many assays. These benefits are used for production of high-quality ELISA kits as well as in research applications now. this can change. The real OD signal is shown without any normalisation. which can really differ from batch to batch or even from well to well. AppliChem now offers a product for use in every research lab in volumes starting as small as 50 ml. This easy to perform process has been an industry standard for thirty years. Saving time and potentially improving the quality of the results of a lab. home-made ELISA are required. 2. because after storage of some days the plates don’t perform as well as before. 2001) or according to other validation strategies. which could potentially falsify interpretation of the results. For stabilisation of a plate one has to incubate with a coating stabiliser solution. A sandwich ELISA with a monoclonal antibody has been done. The positive effects of AppliCoat Plate Stabiliser are shown in Fig. If this plate was stored dry at 4°C the test would correlate to around 2 years of storage. A7099) or BSA based solution (e. but also stabilised. This is a key benefit of AppliCoat Plate Stabiliser. Sometimes however. One can easily coat. A7252) and other blockers. which is called the AppliCoat Plate Stabiliser (Cat. 1. 125 ml and 500 ml.Improving quality & stability of ELISA Using ready-to-use ELISA kits from manufacturers is easy and convenient. most of the antibodies are not active. 3. such as limits of detection are not appropriate. Thus the new workflow in labs has three great advantages: 1. Blocking Buffer III BSA. Preserving correct conformation during storage. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Less work. No. because coating and blocking of many plates can be done in one run instead of doing this work for any new plate again and again. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. It can be used in combination with standard blocking solutions like casein (e. A7708). All those plates are therefore produced in one batch in your lab minimising plate-to-plate variations coming from differences in plate production. Two benefits with one solution When antibodies are coated onto ELISA plates. This stabiliser solution is easy-to-use and has a great advantage compared to almost any stabiliser used in industry. Only around 2–8 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate. Lower variations in measurements from plate to plate as described above.g. Such effects depend on the used antibodies. Even small labs can make use of it. Figure 1 shows a high temperature “stress test” at 37°C for 84 days after application of the stabiliser solution. Why is there such a great difference in the suitability for storage between homemade ELISA and commercial ELISA kits? The reason is that in commercial ELISA kit production the plates are not only blocked after coating. Ready-to-use ELISA kits from good suppliers may be stored for two years at 4°C without any problem. It gives better storage stability for coated antibodies and antigens than most other products do. The result is that most antibodies coated on a plate are unfolded or inactive. the plates can be stored in the fridge for several months.g. And there is a second benefit. block and stabilise as much plates as needed for the next 3 to 6 months. because there is no kit available with the right antibodies or characteristics. One can clearly see the better binding activity when AppliCoat Plate Stabiliser is used. Those plates can be used from time to time for measurements. but when ELISA are validated (e. If there was a way of refolding antibodies and of preserving antibodies from conformational changes during storage. For any researcher this industrial stabilisation technology is made available by AppliChem. It is just as simple as a “second blocking step”.
Then the plates are dried and sealed in plastic or aluminium foil for dry long-term storage Figure 2. B and C show a cceptable stabilization. blocking and stabilization of ELISA plates in a 3 step and in a 2 step process. lates are washed 3 times with 200-300 µl PBS or a ashing Buffer without P W detergents (e. The stabilizers of the second generation A. Here we show the measurements at 40 ng/ml. the shelf life will be around 1 to 3 years. Coating. Calibration curves of an ELISA are shown. Activation of binding capacity by AppliCoat Plate Stabilizer can be seen quite clearly. After stabilization with AppliCoat Plate Stabilizer. Stress test at 45°C with a monoclonal antibody. This needs around 60 to 120 minutes depending on incubator type. 1. 3 the complete calibration curves are shown (only for BSA blocking). emove AppliCoat Plate Stabiliser by suction. Thus if plates should be stored for more than 6 months. that AppliCoat Plate Stabilizer can be combined with different blockers without any problems. Calibration curves at the starting day are identical. dd 200 µl AppliCoat Plate Stabiliser and incubate for 30-90 minutes at apA prox. this unstable antibody shows even after 66 days of incubation at 45°C more than 85% of its activity and therefore an impressively enhanced stability. Figure 3.Figure 1. The solutions are added. incubated and removed again. 20-30°C 5. © 2010 AppliChem • Immunoassay Buffer 19 . Coating as routinely. During this stress test the ELISA after stabilization does not show a decrease in sensitivity even after 84 days of incubation. The used plates. The sensitivity of the assay without stabilization d ecreases rapidly during storage at 37°C. When plates are stored dry. 6. 2. coating concentration. shows the simple three step production process. procedures and buffers have additional impact on achievable stability. which was either blocked only with BSA (red) or blocked with BSA and additionally stabilized with AppliCoat Plate Stabilizer (blue). number of plates in the incubator and speed of air circulation in the incubator. It is also shown. a modern stabilizer of the third generation. 3. Incubate the plates at 37°C R until dry. Blocking as routinely. Figure 4. which can be used for kit production as well as for homemade ELISA. How to use AppliCoat Plate Stabiliser? Figure 1. tore the plate with a lid in the fridge for 3 to 6 months. This antibody showed even after stabilization with carbohydrate containing solutions of the first generation no binding functionality after 24 hours (data not shown). Washing Buffer TrisT- (10x) order number A7137) 4. In fig. a stress test at 37°C or 45°C for storage should be done with the plates to easily measure the shelf life of the plate in a short time. Alternatively plates can S be sealed with plastic or aluminium foil under dryness.g. Achievable shelf life can differ from antibody to antibody. Binding activity of an ELISA at the starting day and after storage for 84 days at 37°C is shown.
Changing the composition has no negative influence on the performance of the products. a mercury-containing molecule which is dangerous for the environment. 20 Immunoassay Buffer • AppliChem © 2010 .c. sterile filtered.less is more! Sometimes. fungi). One of these additives is Thimerosal. FYI: Thimerosal is classified as toxic. For The Sake Of The Environment Many reagents prepared in biomedical research labs are ideal nutrient broths for unwanted germs (bacteria. s. but now we have replaced it by the nontoxic ProClin® 300. compared to ethidium bromide with a lethal dose (mouse. s. For the sake of the environment.) is 9 mg/kg. reagents are either autoclaved.c. Thimerosal is a forbidden additive.) of 110 mg/kg. In some countries. To prevent their growth. For Better Results For Your Safety We would like to keep you healthy so that you stay our customer. Our original immunoasssay buffer contained this chemical too. or antibiotics / antimycotics or toxic substances are added. With CrossDown and all other immunoassay buffers you are even in a better mood and your immunoassays show a better quality. The lethal dose (rat.
80 ng/ml in Antibody Stabilizer-Tris. Many antibodies may be stored even at concentrations as low as e.0125 50 ml 125 ml pH value pH 7. an insoluble precipitate is formed. Avoid freezing * at 2-8°C: 2 years Instructions for use Antibody Stabilizer-Tris is made for long-term storage of proteins and antibodies at 2-8°C in solution. Immediately before use Antibody Stabilizer-Tris should be mixed thoroughly. You can also use Antibody Stabilizer-Tris for storage of coated ELISA plates.1 % ProClin®300 2-8°C. After blocking add Antibody Stabilizer to the coated plates. Stability depends on characteristics and concentration of the used proteins and antibodies. Thus proteins and antibodies are prevented from loosing functionality during storage. ProClin® is a registered trade mark of Rohm and Haas Company. Stability of proteins and antibodies differs significantly from case to case. The user has to test it therefore with its own proteins or antibodies.products Instructions for use Antibody Stabilizer-PBS Stabilization buffer for long-term storage of antibodies and proteins A7148 A7148.2 ± 0. Many antibodies may be stored even at concentrations as low as e. Stability depends on characteristics and concentration of the used proteins and antibodies.2 Preservative contains 0. Thus proteins and antibodies are prevented from loosing functionality during storage. Usually. Just dissolve proteins or antibodies with Antibody Stabilizer-PBS for storage in the refrigerator. Just dissolve proteins or antibodies with Antibody Stabilizer-Tris for storage in the refrigerator.1 % ProClin®300 Storage 2-8°C.2 contains 0. but significant further dilution is possible. Usually.g.0125 50 ml 125 ml pH value Preservative Storage Stability pH 7.g. Components of Antibody Stabilizer conserve the structure of proteins and antibodies in solution.4 ± 0. Avoid freezing * Stability at 2-8°C: 2 years * Upon freezing. Components of Antibody Stabilizer-Tris conserve the structure of proteins and antibodies. Immediately before use Antibody Stabilizer-PBS should be mixed thoroughly.0050 A7135. Stability of proteins and antibodies differs significantly from case to case. Such low concentrations do not require pre-dilutions before every single application. without significant loss of binding activity within the first two years. In this manner treated plates can be stored for a longer time in the refrigerator. 80 ng/ml in Antibody Stabilizer-PBS. After blocking add Antibody Stabilizer-PBS to the coated plates. In this manner treated plates can be stored for a longer time in the refrigerator. but significant further dilution is possible. Antibody Stabilizer-PBS is ready-to-use. The typical concentration of antibodies during storage is 400 to 1000 ng/ml. Antibody Stabilizer-Tris is ready-to-use. You can also use Antibody Stabilizer-PBS for storage of coated ELISA plates. The activity is not significantly reduced! Antibody Stabilizer-PBS is made for long-term storage of proteins and antibodies at 2-8°C in solution. The typical concentration of antibodies during storage is 400 to 1000 ng/ml. Such low concentrations do not require pre-dilutions before every single application. Antibody Stabilizer-Tris Stabilization buffer for long-term storage of antibodies and proteins A7135 A7135. antibodies / proteins are diltued in Antibody Stabilizer-Tris at least by a factor of 1:20.0050 A7148. antibodies / proteins are diltued in Antibody Stabilizer-PBS at least by a factor of 1:20. The user has to test it therefore with its own proteins or antibodies. © 2010 AppliChem • Immunoassay Buffer 21 . without significant loss of binding activity within the first two years.
Blocking Buffer I is characterized by a higher blocking efficiency than most other known blocking reagents. AppliCoat Plate Stabilizer is simply added to the coated microtiter plates.0125 125 ml A7708.1 % ProClin®300 2-8°C. 4a. Moist storage: Cover the plate with adhesive film and store at 2-8°C for up to 3 months.products pH value Preservative Storage Stability AppliCoat Plate Stabilizer Preservative for long-term storage of coated surfaces in immunoassays A7708 A7708. polystyrene beads or glass slides. An additional washing step is not necessary.0050 50 ml A7708. AppliChem A7137). Longer shelf lives have been observed. It is used for long-term storage of precoated plates and other surfaces used in immunoassays.5 ± 0. The assay buffer or the specimen can be added directly onto the sealed plate (solid phase) for use in the assay. Incubation typically takes 60 to 120 minutes. incubator type. the plate can be stored either directly under moist conditions or after drying of the plate or solid phase. Instructions for use 1. This layer is distinguished by good solubility without affecting assays performed at a later date. Figure 1: High temperature “stress test” at 37°C for 84 days after application of the plate stabilizer solution. After incubation of the microtiter plate or solid phase with AppliCoat Plate Stabilizer. Stabilization is similar to a “second blocking step”. which could potentially falsify interpretation of the results. AppliCoat Plate Stabilizer seals by forming a uniform stabilizing layer over antibodies and antigens immobilized on the solid phase. When this plate would have been stored not at 37°C but dry at 4°C the test would correlate to around 2 years of storage Figure 2: Application of AppliCoat Plate Stabilizer. Therefore any assay has to be tested for its individual shelf life. Store the plates sealed in plastic foil or aluminium foil under dry conditions (where required with desiccant) at 2-8°C for 1 to 3 years. AppliCoat Plate Stabilizer seals and stabilizes coated proteins and antibodies. AppliCoat Plate Stabilizer is free of proteins. Apply commonly used coating and blocking procedure for microtiter plate.g. One can clearly see the better binding activity when AppliCoat Plate Stabilizer is used. The values for shelf life of sealed plates are to be used only as a guide.0500 500 ml pH 6. After blocking: Wash 3 times with 200-300 µl PBS or Washing buffer without detergents (e. The true OD signal is shown without any normalisation. AppliCoat Plate Stabilizer is used directly after blocking and washing. Add 200 µl AppliCoat Plate Stabilizer per well and incubate for 15-90 minutes at 20-30°C. After this the ELISA plates are dried in an incubator and can be stored in a fridge for a long time. In the case of strong background problems we recommend the use of AppliChem Blocking Buffer I (A7099) before sealing. 2. Dry storage: Remove the AppliCoat Plate Stabilizer by aspiration. 22 Immunoassay Buffer • AppliChem © 2010 . at 2-8°C: 1 year Improving quality of ELISA measurements and enabling easier workflow in research labs AppliCoat Plate Stabilizer is a ready-to-use reagent for the preservation of immobilized antibodies and proteins. or 4b. The shelf life of the coated molecules is substantially extended to typically 1 to 3 years when stored cool and dry. depending on temperature. but this may not be generalized for all assays. Incubate the plates at 37°C until dry. 3. number of lates and the (active) air circulation in the incubator.2 contains 0.
Blocking Buffer I Background reduction Extremely efficient background reduction – even in critical assays For use in all immunoassays Other blockers Commonly known background reduction Some products only for use in ELISA or Western blotting.Blocking Buffer I Solution for blocking unspecific binding sites for ELISA. validation criteria are met easily Cooling and freezing for long-time storage. Western blotting. for new FDA-guidance for industry) Storage and transportation Ready-to-use Usable with all common detection methods very good results with peroxidases. immuno-PCR. Blocking Buffer I is ready-to-use. protein arrays as well as immunohistochemistry. Repeated freezing and thawing is possible. RIA. no decrease in variations shown Cooling or freezing for long-time storage.g. repeated freeze / thaw cycles are possible at 2-8°C: 6 months Instructions for use Blocking Buffer I saturates free binding capacities on plastic consumables and other surfaces like ELISA plates and blotting membranes. Thus a reduction of unspecific binding on surfaces can be achieved. Efficiency of blocking is significantly improved in comparison to standard blocking procedures by a special production method.0125 125 ml A7099.0050 50 ml A7099. After immobilisation of capture antibody or target protein Blocking Buffer I is applied without dilution in wells or on membranes. Blocking Buffer I can be used in ELISA. negative quenching with fluorescent dyes has to be checked with some products Normal effects on validation. cooled transportation for many products recommended Usability Ease of application Usability with different detection methods Effects on validation (e. EIA & Western Blots A7099 A7099. After blocking the surface has to be washed with Washing Buffer to make it useable for the next working steps. phosphatases and fluorescent labels Positive effects. variations decrease.1 % ProClin®300 –20°C or at 2-8 °C at –20°C: 1 year. which leads to casein molecules with many different molecular sizes.2 contains 0. Incubation time has to be adopted depending on surface characteristics by the user. many different specialised products For some products pre-dilution with other buffers recommended Usability depends on product. repeated freezing and thawing possible.0500 500 ml Composition pH value Preservative Storage Stability low-molecular weight highly purified casein with NaCl and Tween® pH 7.2 ± 0. Immediately before use the buffer should be mixed thoroughly. We recommend blocking over night at 4 °C. EIA. but no cooled transportation needed © 2010 AppliChem • Immunoassay Buffer 23 . background effects are avoide. but in many cases shorter incubation is also promising.
g. A7137).2 contains 0. Blocking Buffer II EGrade saturates free binding sites on the surfaces of e. we strongly recommend using our Casein-based product Blocking Solution I instead (A7099). free of serum and BSA.g Washing Buffer TrisT+ (Art. A7136 or A7150) aspirate Coating Buffer or empty plates by wrapping firmly onto paper cloth. EIA.g. If you have only used Coating Buffer (Prod.0125 125 ml A7516.g. No. If the plate was treated with reagents containing detergents please wash the plate 3 times in a wash buffer free of detergents (e. phosphate-free pH 7. Use Blocking Buffer II EGrade undiluted for incubation of surfaces to saturated free binding sites in non-problematic assays. can lead to satisfactory results. a) Wash membrane 3 times in a wash buffer containing a nonionic detergent. 2. avoiding undesired binding of analytes or detection antibodies to surfaces. If the membrane was treated with reagents containing detergent please wash the membrane 3 times in a wash buffer free of detergents (e. In case satisfying results cannot be obtained with the Blocking Buffer II EGrade . e. microtiter plates 24 membranes © 2010 AppliChem • Immunoassay Buffer 24 . which doesn’t affect high affinity binding in any way. Immuno-PCR peptide-based blocking buffer. CrossDown Buffer acts as a filter for binding. which is used for the immunological detection reaction. serum or tissue specimen. Duration of blocking depends on characteristics of used surface and has to be tested individually.0500 500 ml Solution for blocking unspecific binding sites for ELISA.-Nr.products Composition pH value Preservative Storage Stability Blocking Buffer II EGrade A7516 A7516. Background and nonspecific binding can not only occur at surfaces. Aspirate Blocking Buffer II EGrade or empty plates by wrapping firmly onto paper cloth. 3. The time for blocking depends on the characteristics of the membrane used and has to be tested individually.g Washing Buffer TrisT+ (Prod. or b) add antibody for detection and continue incubation and detection. Incubate membrane in Blocking Buffer II EGrade at room temperature for 1-4 hours or overnight (in most cases one hour is enough). 3. Problems with interactions and cross-reactivities arising from BSA. e. which are present in many blocking solutions. Washing Buffer TrisT-. Prod. Prod. Blocking Buffer II EGrade represents an excellent alternative to BSA-based blocking solutions. Therefore we recommend using CrossDown Buffer (order no. e. It is THE cost-effective alternative to the casein-based Blocking Solution I. if your assay measures analytes in plasma. No. Wash 3 times in wash buffer containing a non-ionic detergent. This leads to significantly reduced background and improved sensitivity of the assay. are avoided by the use of Blocking Buffer II EGrade .1% ProClin® 300 –20°C or 2-8°C Immediately before use the buffer should be mixed thoroughly at -20°C: 1 year. 7158). Suitable for many assays. Immunoassay Buffer • AppliChem © 2010 1. A6485). but depletes nonspecific binding and interference like matrix effects and cross reactivities. Saturation / Blocking 1.g. Duration of blocking can be minimised by shaking the plate at 600-900 rpm. No. but also between antibodies and components of complex specimen. as it is free of serum proteins such as BSA.0 ± 0. Protein Arrays. Western Blots. Blocking Buffer II EGrade is a ready-to-use reagent and applied like common blockers. 2. Washing Buffer TrisT-. repeated freeze / thaw cycles are possible at 2-8 °C: 6 months Blocking Buffer II EGrade is the most economic solution (Economical Grade) for blocking of unspecific binding sites. ProClin® is a registered trade mark of Rohm and Haas Company. Add 200-300 µl Blocking Buffer II EGrade to each well. No. • effective blocking • simple and economically • BSA-free Instructions for use In many assays. In this cases only an assay buffer. Western blotting membranes or slides. A7158). Incubate at room temperature for 1-4 hours or overnight (mostly one hour is quite enough). Blocking Buffer II EGrade prevents nonspecific and unwanted binding to surfaces. microtiter plates (plastic consumables). A7137).
whereas Blocking Buffer III BSA leads to sufficient results in nonproblematic assays. Immuno-PCR as well as protein arrays and immunohistochemistry. Western blotting. Repeated freezing and thawing is possible. We recommend blocking over night at 4 °C.1 % ProClin® 300 –20 °C or at 2-8 °C at –20 °C: 1 year. The incubation time has to be adopted depending on surface characteristics by the user.Blocking Buffer III BSA Solution for blocking unspecific binding sites for ELISA. which doesn’t affect high affinity binding in any way. repeated freeze / thaw cycles are possible at 2-8 °C: 6 months A7252 A7252. In this cases only an assay buffer. Blocking Buffer III BSA is the well-priced alternative to universally applicable and more complex blockers. After immobilisation of capture antibodies or target proteins Blocking Buffer III BSA is applied without dilution in wells or on membranes. After blocking. Thus. can lead to satisfactory results. Immediately before use the buffer should be mixed thoroughly.0500 500 ml Instructions for use Blocking Buffer III BSA saturates free binding capacities on surfaces of plastic consumables and other surfaces like ELISA plates and blotting membranes.-No. Blocking Buffer III BSA is ready-to-use. If a blocker on basis of BSA (bovine serum albumin) is efficient enough for an assay. a reduction of unspecific binding on surfaces can be achieved. EIA. Immuno-PCR. wo T Dream Teams – © 2010 AppliChem • Immunoassay Buffer 25 . the surface has to be washed with Washing Buffer to prepare it for the next working steps. Background and unspecific binding can not only occur at surfaces.4 ± 0. which leads to casein molecules varying in molecular sizes.2 contains 0. Blocking Buffer III BSA is the standard surface blocker for many applications. but also between antibodies and components of complex specimen. CrossDown Buffer has an effect like a filter for binding. A7099). This is achieved by chemical modification from highly purified casein.-No. Efficiency of blocking is significantly improved with Blocking Buffer I in comparison to standard blocking procedures by the special production method of this casein containing reagent. EIA. immunohistochemistry & Western Blots Composition pH value Preservative Storage Stability Standard Blocking reagent with BSA and Tween pH 7. Therefore. A6485) for optimal results in measurements of complex and important specimen. If you find just the same background or unspecific binding in spite of correctly used Blocking Buffer III BSA. we recommend the use of Blocking Buffer I (Prod. you get a maximum of safety and reproducibility. Therefore. but depletes unspecific binding and interference like matrix effects and cross reactivities. but in many cases shorter incubation is also promising. which is used for the immunological detection reaction. we recommend to use CrossDown Buffer (Prod. Blocking Buffer I is suited for blocking of surfaces in all immunoassays.0125 125 ml A7252. Blocking Buffer III BSA can be used for ELISA.
1200). heat to 60 -65°C in a water bath or microwave oven. 6. and for Western blots. 600 mM NaCl). Southern blots. and add successive squares at 10minute intervals. 8. When immunoassays and hybridization assays. and stir well until dissolved. Label each square with a ball point pen in 10 minute increments (60. Note Do not use the Blocking Reagent CA in PBST for alkaline phosphatase conjugate dilutions Nucleic Acids For hybridization applications add 0. Rinse in TBST. 5. and before incu-bation in any enzyme-conjugate solution (e. 2. Incubate on shaker for 1 hour.2 % (w/v) Blocking Reagent CA into TBST or PBST. 10 minutes each time. Cut 7 small squares of nitrocellulose or other suitable membrane. 40. 3. 7. The Blocking Reagent CA dissolves to give a milky solution. PBST or Tris-Saline buffer (100 mM Tris-Cl pH 7.5. A3417. Streptavidin-HRP.2 % (w/v) Blocking Reagent CA to Tris-Saline buffer (100 mM Tris-Cl pH 7. Blocking Reagent CA is used to "block" unbound sites left after immobilization of the specific protein or after the hybridization with non-radioactive probe. and stir well until dissolved. 600 mM NaCl). Dilute the secondary antibody or streptavidin (HRP-conjugated) in Blocking Reagent CA solution. there is nonspecific binding resulting in high background. 50. or Northern blots are performed. Wash all squares in TBST. PBST or Tris-Saline buffer three times.g.0010 Storage Room Temperature The Blocking Reagent CA is used in hybridization and detection procedures using non-radioactive nucleic acid probes. Optimization of Time Required for Blocking with the Blocking Reagent CA 1. The Blocking Reagent CA improves sensitivity and reduces background. In order to reduce the nonspecific binding. The Blocking Reagent CA dissolves to give a clear solution. Western blots. Detect with the Chemiluminescent Detection Kit for horseradish peroxidase (Order-No. such as dot blots. 20. and one without blocking. Streptavidin-AP). add 0. 9. heat to 75-80 °C in a water bath or microwave oven. Select the incubation time that gives the lowest background.products Blocking Reagent CA Blocking Reagent for Hybridization Assays and Western Blots A3409. 10). 26 Immunoassay Buffer • AppliChem © 2010 . Note Nonfat dry milk inhibits the streptavidin-biotin interaction due to its content of biotin! Procedure Proteins For blotting applications such as Western blots and dot blots.5. 4. Use for blocking and for dilutions of antibodies. Use for blocking after the wash steps. Evaluate background intensity in each square. Place the first square (60) in a few ml of Blocking Reagent CA solution. 30.
EIA.0125 125 ml Composition pH value Preservative Storage Stability PBS-based 10x stock solution pH 7. Consequently any user should optimise its own incubation procedure. © 2010 AppliChem • Immunoassay Buffer 27 . thus having an effect on immobilisation. 3 months Instructions for use Coating Buffer PBS pH 7. Use the working solution the same day. The typical concentration range for standard ELISA is between 0. The stock solution is diluted 1:10 with deionized water to get the working solution.4 must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution.6 may have advantages for immobilisation. Thus coating buffer is safe and easy useable for many applications –20°C or at 2-8°C at –20°C: 1 year at 2-8°C: min.6 ± 0.4 Coating buffer for capture-antibodies and proteins on surfaces A7136 A7136.2 Buffer is delivered without any preservatives.5 µg/ml to 2 µg/ml for capture antibodies. Crystals of salt can precipitate during storage at 2-8 °C or after freezing. for others Coating Buffer C pH 9.6 Coating buffer for capture-antibodies and proteins on surfaces Composition pH value Preservative Storage Stability A7150 A7150.Coating Buffer C pH 9.6 is made for adsorptive immobilisation of proteins and antibodies on plastics surfaces (for example microtiter plates) or other protein binding surfaces. thus having an effect on immobilisation.5 µg/ml and 2 µg/ml for capture antibodies.0125 125 ml Carbonate-based 10X stock solution pH 9. EIA. For some proteins or antibodies Coating Buffer PBS pH 7. The proteins or antibodies for immobilisation are diluted in this working solution and used after mixing. RIA and protein arrays as well as immuno-PCR.2 Buffer is delivered without any preservatives. Therefore Coating Buffer C must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. because some preservatives can interfere with the process of coating. Coating Buffer PBS pH 7. This leads to dissolving of salt after shaking.4 is better. Depending on the surface as well as on proteins or antibodies the useful incubation times can differ.4 ± 0. The pH-value can have an influence on the steric structure of proteins or antibodies. 3 months at 2 -8°C: 1 month Instructions for use Coating Buffer C pH 9. RIA and protein arrays as well as immuno-PCR. Depending on the surface as well as on proteins or antibodies the useful incubation times can differ.4 is made for adsorptive immobilisation of proteins and antibodies on plastics surfaces (for example microtiter plates) or other protein binding surfaces. Thus coating buffer is safe and easy useable for many applications –20 °C or at 2-8°C Use working solution immediately! at –20°C: min. because some preservatives can interfere with the process of coating.6 is advantages for immobilisation. Use the working solution the same day. The typical concentration range for standard ELISA is 0. For an optimised procedure for a newly developed immunoassay we strongly recommend testing of both Coating Buffers in comparison. This leads to dissolving of salt after shaking. Applications are for example ELISA. The proteins or antibodies for immobilisation are diluted in this working solution and used after mixing. Therefore Coating Buffer PBS pH 7. Applications are for example ELISA.4 is better. Crystals of salt can precipitate during storage at 2-8 °C or after freezing. The pH-value can have an influence on the steric structure of proteins or antibodies. for others Coating Buffer C pH 9. Consequently any user should optimise its own incubation procedure. The stock solution is diluted 1:10 with deionized water to get the working solution. For some proteins or antibodies Coating Buffer PBS pH 7. For an optimised procedure for a newly developed immunoassay we strongly recommend testing of both Coating Buffers in comparison.
2 ± 0. Therefore Literatur  Miller. W. unspecific binding and matrix effects in immunoassays like ELISA. ready-to-use contains 0. We strongly recommend to test the effectiveness of CrossDown Buffer for a certain application. J. ***Alexa Fluor Dye is a registered trade mark of the company Molecular Probes. Clin. J. P. multianalyte immunoassays and immunohistochemistry – depending on the characteristics of the assay type and the used antibodies.and middle-affinity binding. Valdes. depending on the recommendation of the data sheet of the antibodies. But in this case also the unwanted bindings or cross-reactivities can partly occur again. G. J.2 Phosphat-free. Polyclonal antibodies normally contain low.and middle-affinity binding will be still reduced by CrossDown Buffer. Dilution of antibodies Antibodies can be diluted with CrossDown Buffer in a user-defined manner... (2004) Clinical Laboratory International 28. J.. CrossDown Buffer is not suited as a sample buffer for electrophoresis. Hoheisel. W.0125 125 ml A6485.Jr. A7099). EIA. 32.G. protein arrays.J. (2000) Electrophoresis 21.0050 50 ml A6485. Western blotting. 97-107  Wood.J. 1708-1709 28 Immunoassay Buffer • AppliChem © 2010 . 16. 205. In case that CrossDown is to “active” (i. Appearance of signal reduction In some cases a smooth reduction of the wanted signal can be observed.D.J. Although CrossDown Buffer is used as an assay buffer it is necessary to saturate surfaces like ELISA-wells or membranes with a blocking agent.N.(Molecular Probes) fluorescence dyes. Invest.products CrossDown Buffer Immunoassay buffer for minimisation of unspecific binding. 1123-1144  MacBeath.and middle-affinity binding components. C. (2002) Nat. Components of immunoassays – as well as of CrossDown Buffer– may quench the fluorescence of fluorescein dyes. A7099). (2003) Clinical Chemistry 49 (10). For blocking of surfaces we recommend Blocking Buffer I (Order No. Recognit. Geurts-Moespot. 14-17  Kusnezow. cross-reactivities and matrix effects pH-Value Stabilizer Storage Stability pH 7. (2).0500 500 ml Instructions for use The newly developed CrossDown Buffer lowers cross reactivities. (1999) Clinical Chemistry 45 (7). F Dilution of the specimen Standards and specimen for ELISA and protein arrays can be diluted with CrossDown Buffer at 1:2 or higher. Standards and specimen should be treated strictly the same way. Lab. Mix the buffer thoroughly immediately before use.J.F. 105-112  Kricka. Sweep. CrossDown Buffer reduces low. We recommend the use of Blocking Buffer I (Order No. L. depending on the chosen dilution with water. Genet. physiological buffers like PBS or Hepes can be used for diluting CrossDown.G. R. *Oyster is a registered trade mark of the company Denovo Biolabels. Alternatively. repeated freeze/thaw cycles possible at 2-8°C: 6 months A6485 A6485. immuno-PCR.. This is the same for primary and secondary antibodies. Suppl..and middle affinity antibodies (also monoclonal antibodies) a pre-dilution of CrossDown Buffer with salt-free water can be useful to get the previously seen signal. (2003) J. J. 526-532  Miller. Grebenchtchikov N.1 % ProClin®300 (ProClin® is a registered trademark of Rohm and Haas) –20°C or 2-8°C at –20°C: 1 year. CrossDown Buffer is not suitable for blocking of surfaces. CyDye**(Amersham) or Alexa***.(Denovo Biolabels). In the case of low. CrossDown in FACS analysis CrossDown buffer can replace the normally used FACS analysis assay buffer and is applied like the original assay buffer. Examples of use ELISA dilution buffer for specimen and for the detection antibodies Western blotting dilution buffer for primary and secondary antibodies Immunohistochemistry dilution buffer for primary and secondary antibodies Protein arrays dilution buffer for specimen and for the detection antibodies we recommend the use of Oyster*. Unwanted low. reduction of the specific signal too). CrossDown Buffer can be used additionally as a washing buffer – especially in delicate or interference-sensitive assays like immuno-PCR. **CyDye is a registered trade mark of the company Amersham Biosciences. (1991) Scand. W. CrossDown Buffer is used instead of a sample buffer or antibody dilution buffer for the immunological reaction. 165-176  Patton. (1992) J Clin Immunoassays 15.e. That means that by the use of lowand middle-affinity antibodies or polyclonal antibodies a smooth reduction of signals can appear. 942-956  Span. In the case of polyclonal antibodies a moderate increase of the concentration of the antibody can lead to the previously seen signals. Mol. the most convenient way is to dilute the buffer with the original assay buffer (dilution 1 : 2 to 1 : 10).
The specific. The primary cause of the so-called matrix effect is based on unwanted. unspecific binding and matrix effects in immunoassays like ELISA. Can CrossDown be applied together with fluorescent dyes. you have to keep in mind that CrossDown only supports high affinity binding. preventing the binding of the antibody to its target. oyster dyes). In case of polyclonal antibodies. The optimal conditions and ratios should be determined on a case by case basis. low-affinity binding of matrix components (e. protein arrays. Antibodies may be masked by matrix components as well.g. Western blot membranes or ELISA plates)? The buffer can be frozen and thawed several times without a problem. There are positive results in protein chip applications. Polyclonal antibodies are a mixture of antibodies with different affinities. serum proteins in samples from human serum) to analytes or antibodies. an increased signal was observed. Cy5. No! CrossDown Buffer is not suitable for blocking of surfaces.FAQ What's so special about CrossDown? an antibody in an assay has to be adjusted very much depends on the quality of the antibody The newly developed CrossDown Buffer lowers cross reactivities. but used for dilution of the sample and/or assay antibodies instead. Assuming nonspecific binding or cross reactivity of the detection antibody. Western blotting. human serum or plasma) and the detection antibody are diluted with CrossDown Buffer. Users report an increased enzyme activity. Is the buffer used diluted or undiluted? CrossDown Buffer is a ready-to-use solution. After thawing. e. © 2010 AppliChem • Immunoassay Buffer 29 . In some applications using a few cyan-dyes (e. while the effect of low affinity antibodies will be minimized. it may make sense to dilute CrossDown Buffer with water or physiological buffer. For blocking of surfaces we recommend Blocking Buffer I (Order No. when labeled detection antibodies were incubated with CrossDown Buffer. CrossDown Buffer prevents masking and support binding of antibody and analyte. CrossDown prevents this masking and reduces masking already present. Increasing the antibody concentration increases the concentration of high affinity antibodies as well. A7099). multianalyte immunoassays and immunohistochemistry. In competitive assays and some applications in immunohistochemistry. In fact. Cy3.g. Do I have to consider whether polyclonal or monoclonal antibodies are diluted with CrossDown? No! CrossDown can be used in combination with both types of antibody preparations. depending on the source of the interference. Whether the concentration of Do buffer components influence color reactions? The enzymatic activity of alkaline phosphatase or peroxidase are not negatively influenced by CrossDown Buffer. while unwanted binding of the antibody is prevented. Why does CrossDown Buffer assist in reducing matrix effects? How do I use CrossDown Buffer? CrossDown Buffer is used instead of sample buffer or antibody dilution buffer for the immunological reaction. The sample (e.g. Therefore it may be necessary to increase the concentration of a poylclonal detection antibody when used with CrossDown Buffer. in protein chip applications? Yes. high affinity binding of antibody to analyte stays. The sample or the detection antibody can be diluted directly in CrossDown Buffer.g.g. Low affinity binding is suppressed. immuno-PCR. dilution with CrossDown Buffer is recommended. EIA. May I freeze-thaw the buffer several times? Does CrossDown Buffer replace a blocking buffer for surfaces (e. the buffer has to be mixed thoroughly before application to guarantee uniform distribution of the components. The analyte may be masked by proteins or other components of the sample matrix. the opposite is the case.
negative quenching with fluorescent dyes has to be checked with some products Some products recommend pre-dilutions with other buffers No positive effects on validation. repeated freezing and thawing possible. No effect on matrix effects and other cross-reactivities Cooling or freezing for most products necessary. variations decrease. most products recommend cooled transportation Positive effects only if HAMAs are inside the samples. many different specialised products Usability depends on product. phosphatases and fluorescent labels Ease of application Ready-to-use Stabilisation of antibodies Assay antibodies are stabilised in CrossDown Buffer. for new FDA-guidance for industry) Storage and transportation using CrossDown Buffer?? HAMA-Blocker Minimisation only of interference derived from HAMAs – (Human Anti Mouse Antibodies) – All other interference effects lead to wrong results! Effects only if background comes from HAMAs Increase in reliability only when specimen / samples include HAMAs For use only for human specimen/ samples Usability depends on product.g. but no cooled transportation needed No effects on stability of assay antibodies Effects on validation (e. matrix effects or unspecific binding of assay components Minimisation of background Increase in reliability guarantees better results by avoiding interference For use in all immunoassays Antibody Diluent Background Quality of results No effects No positive effects on reliability Usability Usability with different detection methods Usable with all common detection methods. some only for use with peroxidases others only for use with phosphatases. validations can be passed successful and easy Cooling and freezing for long-time storage. some only for use with peroxidases others only for use with phosphatases. even storage of antibodies in CrossDown is possible Positive effects. Then false results can be avoided. interference like matrix effects or cross-reactivities lead to high variations or false results Cooling or freezing for long-time storage. negative quenching with fluorescent dyes has to be checked with some products Some products recommend pre-dilutions with other buffers No effects on stability of assay antibodies No minimisation of interference Some products only for use in ELISA or Western blotting. cooled transportation needed 30 Immunoassay Buffer • AppliChem © 2010 .Why CrossDown Buffer Interference effects Minimisation of interference – regardless whether cross-reactivities. false results are avoided. very good results with peroxidases.
CrossDown Peroxidase-Stabilizer is a combination of CrossDown Buffer and a new stabilizer for stabilizing antibodies and peroxidase. Unfortunately this significantly increases the risk of interference. CrossDown Buffer reduces low and medium-affinity binding. Thus convenience and reliability are no longer incompatible. That means that by using low. If CrossDown Peroxidase-Stabilizer is used for immunodiagnostic kits.0500 500 ml pH value Preservative Storage Stability pH 7.2 ± 0. For this reason reliability suffers in favour of user-friendliness.CrossDown Peroxidase-Stabilizer Assay-diluent for one-step incubation assays peroxidase conjugate stabilizer l substitute for HAMA blocker l l products A8625 A8625. 1). Stabilization of a HRP-antibody conjugate by a competitor's enzyme stabilizer product was compared to stabilization by CrossDown Peroxidase-Stabilizer (see fig. which apply. Testing CrossDown Peroxidase-Stabilizer with any new assay is recommended. Do NOT freeze. Typical concentrations of conjugates are ranging from 40 to 500 ng/ml. Observation of signal reduction: In some cases a slight reduction of the wanted signal can be observed.0050 50 ml A8625. the shelf life has to be tested according to the current European directive for invitro-diagnostics or according to corresponding rules. Conjugates can be diluted directly in CrossDown Peroxidase-Stabilizer to the final concentrations.and medium-affinity binding components. 24 months Description In one-step incubation assays the specimen is incubated directly with the detector-conjugate saving additional washing steps. Therefore every conjugate has to be tested for its shelf life in CrossDown Peroxidase-Stabilizer. Figure 1: Shelf life of HRP-antibody conjugate bound to the analyte is measured as peroxidase signal in an ELISA and given as % value of freshly diluted HRP-antibody conjugate. Polyclonal antibodies normally contain low. In the case of polyclonal antibodies a moderate increase of the concentration of the antibody can lead to the previously seen signals.0125 125 ml A8625. Detector-conjugate concentration was 40 ng/ml. CrossDown Peroxidase-Stabilizer enables assays with the highest reliability and robustness by minimising interference.or Streptavidin-peroxidase-conjugates. This can be antibody-peroxidase-conjugates as well as Neutravidin. Stability data of one peroxidase-conjugate cannot be transferred to other conjugates.and medium-affinity detector-antibodies or polyclonal detector-antibodies a smooth reduction of signals can appear. Immediately before use the buffer should be mixed thoroughly. Therefore conjugates can be stored directly in very low ready-to-use concentrations. This makes CrossDown Peroxidase-Stabilizer the ideal solution. HRP – long-term stabilization of peroxidase: CrossDown Peroxidase-Stabilizer contains stabilizing components for antibodies and for peroxidase. The CrossDown Buffer effect: CrossDown Buffer was specially developed to minimise cross reactivities. if you want to store peroxidase-conjugates for a long period in a ready-to-use solution.2 contains 0. Instructions for use CrossDown Peroxidase-Stabilizer is a dilution buffer for long-term storage of peroxidase-conjugates. This is very convenient and enables for very fast assay protocols in comparison to sequential procedures. Dilute the conjugates directly in CrossDown Peroxidase-Stabilizer. Interference derived from HAMA (human anti-mouse antibodies) rheumatoid factors or other endogenous interfering factors can be reduced significantly. CrossDown Peroxidase-Stabilizer provides the best long-term stabilization of the HRP-antibody conjugate. Therefore you get long-term stabilization as well as the CrossDown Buffer effect in one solution. © 2010 AppliChem • Immunoassay Buffer 31 . May form a precipitate.1 % ProClin®300 (ProClin® is a registered trademark of Rohm and Haas) 2-8°C. CrossDown Peroxidase-Stabilizer solves this problem. At the same time CrossDown Peroxidase-Stabilizer is a premium stabilizer for peroxidase conjugates. matrix effects and problems due to HAMAs (human anti-mouse antibodies) and rheumatoid factors. nonspecific binding and matrix effects in immunoassays.
in which HRP is the detector enzyme. even when these concentrations are very low. This makes Peroxidase-Stabilizer the ideal solution.products Peroxidase-Stabilizer pH value Preservative Storage Stability pH 7. Typical concentrations of conjugates are ranging from 40 to 500 ng/ml. Stability data of a peroxidase-conjugate cannot be transferred to another conjugate. Peroxidase-Stabilizer maintains the activity of conjugates of antibodies and of Neutravidin conjugated to HRP. HRPconjugate of the antibody was stabilized either by a so far leading enzyme stabilizer product or by Peroxidase-Stabilizer. If you have background issues or observe false-positives – for example derived from cross reactivities. which apply. it is especially important to ensure adequate stability.0050 50 ml l protection and stability of your HRP-conjugate for years A8647. Immediately before use the buffer should be mixed thoroughly. After 143 days incubation at 45°C with Peroxidase-Stabilizer the monoclonal antibody conjugate retained around 80 % enzymatic HRP activity.0125 125 ml l Peroxidase-Stabilizer maintains enzyme activity A8647. Instructions for use Conjugates can be diluted directly in Peroxidase-Stabilizer in its final concentration. 1). This saves pre-dilution steps before doing an assay. Peroxidase-Stabilizer is designed for troublefree assays without interference. Peroxidase-Stabilizer can be used directly as assay buffer in immunoassays. Therefore it can be used in any assay.2 contains 0.2 ± 0. May form a precipitate. 24 months A8647 dilution buffer and storage solution for peroxidase-conjugates for an outstanding shelf-life of the conjugates A8647. matrix effects or HAMA’s – we recommend using CrossDown-Buffer or CrossDown Peroxidase-Stabilizer rather than Peroxidase-Stabilizer. if you want to store peroxidase-conjugates for a long period in a ready-to-use solution. Figure 1: Shelf life of HRP-antibody conjugate bound to the analyte is measured as peroxidase signal in an ELISA and given as % value of freshly diluted HRP-antibody conjugate. Percent retained activity was determined by comparing the activity of the HRP-antibody conjugate bound to the analyte. Peroxidase-Stabilizer – long-term stabilization of peroxidase: Peroxidase-Stabilizer contains stabilizing components for antibodies and for peroxidase. If Peroxidase-Stabilizer is used for immunodiagnostic kits.0500 500 ml l Peroxidase-Stabilizer maintains functionality of HRP-conjugate l conjugate may be stored at very low final concentration – no pre dilution steps Description Stabilization of all active components is very critical to get reliable results with immunoassays. Dilute the conjugates directly in PeroxidaseStabilizer. Suitability of Peroxidase-Stabilizer for a specific assay has to be tested by the user. 32 Immunoassay Buffer • AppliChem © 2010 . Peroxidase-Stabilizer works very well as a stabilizer. This is a very good stability and therefore PeroxidaseStabilizer gives an optimum product safety for HRP containing immunoassays! Testing Peroxidase-Stabilizer with any new assay is recommended. This shows a shelf life at 4°C with approximately 80% enzymatic activity of more than 6 years by conservative correlation according to Arrhenius. When assays are stored for long-term before use.1 % ProClin®300 2-8°C. This enables any user to store HRP conjugates directly in ready-to-use concentrations. thereby producing more consistent results with your diagnostic test kit. Detector-conjugate concentration was 40 ng/ml. Therefore every conjugate has to be tested for its shelf life in Peroxidase-Stabilizer. Stress tests were performed at 45°C over a period of up to 143 days. Peroxidase-Stabilizer decreases the variability often caused by changing shipping and storage conditions. the shelf life has to be tested according to the current European directive for invitro-diagnostics or according to corresponding rules. Stability test Stability „stress“ tests were performed with Peroxidase-Stabilizer (PS) using a monoclonal antibody conjugate (see fig. Peroxidase-Stabilizer can be easily incorporated into most assay protocols by substituting it for your conjugate diluent. Do NOT freeze. Additionally it is a very good assay buffer.
can be diluted and used in Sample Buffer T+ without problems. we recommend the use of CrossDown Buffer (Artikel-Nr.0500 500 ml Composition pH value Preservative Storage Stability detergent-free. phosphate-free pH 7. It is necessary to treat standards and specimen in the same way! In case you observe increased background or unwanted matrix effects in the presence of Sample Buffer T+. because Sample Buffer T. EIA or Western blotting as well as for immuno-PCR or protein arrays. Sample Buffer T+ can be used for ELISA. repeated freezing and thawing cycles possible at –20 °C: 1 year . Repeated freezing and thawing is possible.0050 50 ml A7134. Sample Buffer T+ is not for use as an electrophoresis buffer.without problems. Immediately before use the buffer should be mixed thoroughly. coupled to alkaline phosphatase or peroxidase.0125 125 ml A7134. Sample Buffer T+ is ready-to-use. Primary and secondary antibodies can be diluted directly in Sample Buffer T-.is ready-to-use. because Sample Buffer T+ is free of phosphate.2 contains 0.0050 50 ml A7101. Immediately before use the buffer should be mixed thoroughly.2 ± 0.1 % ProClin®300 –20 °C or at 2-8 °C. coupled to alkaline phosphatase or peroxidase. Specimen with the analyte – as well as the detecttion antibody – is diluted in Sample Buffer T+ and then used in the assay. A6485) instead of Sample Buffer T+. at 2-8 °C: 6 months Instructions for use Sample Buffer T. © 2010 AppliChem • Immunoassay Buffer 33 . Thus it is for use especially in immunohistochemistry. A6485) instead of Sample Buffer T-.2 contains 0.is a dilution buffer for specimen and antibodies für direct use in immunoassays. repeated freezing and thawing cycles possible at –20 °C: 1 year . In case you observe increased background or unwanted matrix effects in the presence of Sample Buffer T-.is free of phosphate. we recommend the use of CrossDown Buffer (Artikel-Nr.Sample Buffer TSample buffer and dilution buffer for antibodies for use in immunoassays and immunohistochemistry A7101 A7101. Sample Buffer T+ Sample buffer and dilution buffer for antibodies for use in immunoassays A7134 A7134.2 ± 0. Sample Buffer T. at 2-8 °C: 6 months Instructions for use Sample Buffer T+ is a dilution buffer for specimen and antibodies für direct use in immunoassays.0500 500 ml Composition pH value Preservative Storage Stability contains Tween® pH 7. Repeated freezing and thawing is possible. where detergents sometimes can make problems in detection.0125 125 ml A7101.without detergents contains neither Tween® nor other detergents. Sample Buffer T. Antibodies.1 % ProClin®300 –20 °C or at 2-8 °C. Antibodies. can be diluted and used in Sample Buffer T.
8 ± 0. For this purpose gently shake the membrane in Stripping Buffer I for 30-60 minutes at room temperature.0050 50 ml A7140. primary and secondary antibodies from Western blotting membranes. It should be used.0125 125 ml Instructions for use Ready-to-use Stripping Buffer I removes reaction solution.2 ± 0. EIA. This leads to dissolving of salt after shaking.2 contains 0. The stock solution is diluted 1:10 with deionized water to get the working solution.2 contains 0. Crystals of salt can precipitate during storage at 2-8 °C or after freezing. After stripping the membrane can be used for a second detection (reprobing) with the same or other antibodies.(10X) Washing buffer for use in ELISA.is made especially for immunohistochemistry. contains no detergents pH 7. 34 Immunoassay Buffer • AppliChem © 2010 .1 % ProClin®300 –20 °C oder bei 2-8°C Use working solution immediately! at –20 °C: 1 year at 2-8 °C: 6 months Instructions for use This Washing Buffer TrisT. Washing Buffer TrisT. Incubation times are depending on characteristics of the used antibodies. which have to be adopted by the user. Use the working solution the same day.1 % ProClin®300 –20 °C or at 2-8 °C at –20 °C: 1 year at 2-8 °C: 6 months A7140 A7140. The membrane is incubated in a vessel with Stripping Buffer I for removing the antibodies.products Stripping Buffer I Stripping buffer for Western blots for multiple reprobing Composition pH value Preservative Storage Stability doesn't contain β-mercaptoethanol and DTT pH 2. After stripping. the wash buffer should not contain phosphates.0500 500 ml Composition pH value Preservative Storage Stability Tris-based 10X buffer with 350 mM NaCl. Important: The designated incubation conditions are standard values. Therefore Washing Buffer TrisT. whenever detergents can make problems in detection.must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. the membrane has to be washed with Washing Buffer and can be used for a second detection. If you detect with alkaline phosphatase. Western blotting and immunohistochemistry A7137 A7137.
Western blotting as well as immuno-PCR. This leads to dissolving of salt after shaking.1 % ProClin®300 bei –20 °C or at 2-8°C Use working solution immediately! at –20 °C: 1 year at 2-8 °C: 6 months Instructions for use Washing Buffer TrisT+ is used for many immunoassays. EIA and Western blotting A7158 A7158. protein arrays and multianalyte immunoassays. Application fields are ELISA.Washing Buffer TrisT+ (10X) Washing buffer for use in ELISA.without Tween or other detergents (Product No. Caution Make sure that you have checked the specifications and instructions of your washer! Crystals of salt can precipitate during storage at 2-8 °C or after freezing. Use the working solution the same day. A7137).0500 500 ml Composition pH value Preservative Storage Stability Tris-based 10X buffer. which are able to interfere with the assay. 35 . EIA. Therefore Washing Buffer TrisT+ must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. contains Tween® and 350 mM NaCl pH 7.2 contains 0. Washing steps are needed to remove unbound and excessive components. Especially for use in immunohistochemistry we offer Washing Buffer TrisT.2 ± 0. It is applicable in automatic plate washers depending on salt tolerance of the washer. RIA. The stock solution is diluted 1:10 with deionized water to get the working solution.
ISBN 0-08-044526-8 Werner Luttmann. No.45 µm Reprobe Nylon Positively charged Transfer membrane 0.22 µm Transfer membrane Pure Nitrocellulose unsupported 0.45 µm Transfer membrane Pure Nylon Neutral Transfer membrane 0. Kai Bratke.) Immunoassays 2007 Elsevier Spektrum Akademischer Verlag (ISBN 3-8274-1636-1) german language David Wild (Ed. Eur. Raem & Peter Rauch (Hrsg. No. No. Oxford 2005.. A5237 A5242 A5250 A5239 A4399 A5248 A5255 A5243 Further reading Arnold M. 3rd Edition. Boston. Spektrum Akademischer Verlag. Auflage. 2. A0845 A6588 A2248 A3792 A2250 A4970 A1693 A3004 A0830 A2260 A2160 A2159 Prod. Michael Küpper.45 µm Brochure Transfer Membranes Other Related Products Chemiluminescence Detection Kit for Horseradish Peroxidase Peroxidase fom horseradish EIA and Immunology Grade I Peroxidase fom horseradish EIA and Immunology Grade II Sodium azide pure Streptavidin ultrapure Thimerosal BioChemica Prod.45 µm (30 cm x 3 m) PVDF-Star Transfer membrane 0.Related products Blocking Reagents Albumin acetylated Albumin Fraction V (pH 7. (ISBN 3-8274-1730-9) german language 36 Immunoassay Buffer • AppliChem © 2010 .): The Immunoassay Handbook. A3417 A3615 A3771 A1430 A1495 A1278 Prod. Daniel Myrtek: Der Experimentator: Immunologie.Solution (50X) BioChemica Denhardt's powder mixture (for 50X stock solution) Dextran sulfate 500 sodium salt BioChemica Dextran sulfate 500 sodium salt Molecular biology grade Gelatin powdered pure Ph.22 µm (30 cm x 3 m) Pure Nylon Neutral Transfer membrane 0.45 µm Transfer membrane Pure Nitrocellulose unsupported 0.22 µm Transfer membrane Reprobe Nitrocellulose supported 0. Elsevier Science Publishing Company. Heidelberg 2006. Amsterdam.0) Blotting grade Denhardt's . NF Heparin sodium salt Nonfat dried milk powder Polyvinylpyrrolidone (K90) Molecular biology grade Salmon sperm DNA sodium salt Salmon sperm DNA sodium salt (sonified) Transfer membranes Reprobe Nitrocellulose supported 0.
antibiotics for the treatment of infected cells. Read more about the usage of our markers in our new brochure. AppliChem offers the full range of lyophilized DNA markers. Detergents Detergents are more than just air bubbles. They do influence the growth. As diverse as the research objects and techniques are as diverse are the detergents available. Several aspects have to be taken into account. however. and cleansing reagents for CO2-incubators and waterbaths to prevent infection. The properties of e. Biological buffers are an essential part of each experiment.derman! . even though it often goes unnoticed since no cloudiness appears in the cell culture. DNA and protein analysis. der Biotechnologie Safety First: Banish Mycoplasma The contamination of cells with mycoplasma is a very common problem. AppliChem offers a PCR-based test kit to test for infection. Mycoplasma are small and may pass the filtration units applied for preparing cell culture media. The advantage of lyophilized marker is their outstanding stability. AppliChem‘s brochure “Biological Buffers” imparts basic knowledge on the criteria for selecting the right buffer and requirements of buffers. morphology and survival of the host cells. 5/08 Forschst Du noch oder denkst Du schon? Hirnwelt Faszinierende Einblicke Medienwelt Flimmernde Realität Unterwelt Erschreckende Dimensionen Get Appli phantastic customer ma Chem‘s gazine available in English and free of charge: Ge service@applichem. affecting the functioning of the system under investigation.applichem. The long shelf life of more than 5 years is achieved by deproteinization and subsequent lyophilization.com Gel Electrophoresis Size Marker Ready-to-use DNA size marker and protein size marker for gel electrophoresis are available from many sources. without. Biological Buffers Many biochemical processes are markedly impaired by even small changes in the concentration of free H+ ions. It is therefore usually necessary to stabilise the H+ concentration in vitro by adding a suitable buffer to the medium. 22 protocols and all types of membranes – all from one source.g. when planning an experiment. AppliChem also provides our customers with tried and proven protocols developed to obtain consistent reproducible and dependable results when used with AppliChem membranes.infotaining Get your free copy – www. Transfer Membranes AppliChem supplies a range of transfer membranes designed and tested specifically for RNA. SDS and octylglucoside are so different that you hardly may exchange them for the identical experiment. resulting in a strong influence on the test system and the results obtained. Think! für Wissbegierige Von Wissenschaftlern und Pharmaforschung in der Chemie. Besides these standard products. Why do you apply SDS in gel electrophoresis but not dodecylmaltoside? Because your colleagues did it all the time before? AppliChem‘s brochure „Detergents“ will help you selecting the best detergent for your assay.
applichem.A71.com .E 4t Matthes + Traut · Darmstadt There is another top address in Darmstadt: AppliChem GmbH Ottoweg 4 D .64291 Darmstadt Phone +49 6151 9357-0 Fax +49 6151 9357-11 08/2010 eMail email@example.com internet www.
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