Thermo Scientific Pierce Crosslinking Technical Handbook

Table of Contents
Books and Free Technical Handbooks Crosslinkers at a Glance Appendix I - Structures Appendix II - Online Interactive Crosslinker Selection Guide Appendix III - Glossary of Crosslinking Terms 23 24-29 30-43 44 45

Introduction How to Choose a Crosslinker Crosslinking Applications Label Transfer Reagents Sulfo-SBED Reagent Mts-Atf-Biotin Reagents Heavy/Light Crosslinker Pairs Single-step vs. Multi-step Reactions Homo- and Heterobifunctional Crosslinkers Bioconjugate Toolkit Controlled Protein-Protein Crosslinking Kit Activated Dextran Coupling Kit Crosslinker Reactivities Amine-reactive Chemistries N-Hydroxysuccinimide-Esters Sulfhydryl-reactive Chemistries Pyridyl Disulfides Carbonyl-/Glyco-reactive Chemistry Carboxyl-reactive Chemistry Nonspecific Chemistries

1-2 2 3-13 8 9 12 13 14-16 14 15 16 16 17-22 17 17 18 19 19 19 20

Patent Index Sulfo-SBED Biotin Label Transfer Reagent is protected by US Patent 5,532,379. Slide-A-Lyzer MINI Dialysis Unit Technology is protected by US Patent 6,039,871.

Introduction What is crosslinking?
Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. Because of the availability of several chemical groups in proteins and peptides that may be targets for reactions, proteins and peptides are readily conjugated and otherwise studied using crosslinking methods. Crosslinkers also are commonly used to modify nucleic acids, drugs and solid surfaces. Crosslinking reagents have been used to assist in determination of nearneighbor relationships, three-dimensional structures of proteins, solid-phase immobilization, hapten-carrier protein conjugation and molecular associations in cell membranes. They also are useful for preparing antibody-enzyme conjugates, immunotoxins and other labeled protein reagents.
Conformational changes of proteins associated with a particular interaction may be analyzed by performing crosslinking studies before and after the interaction occurs. Comparing crosslinkers with different arm lengths for success of conjugation can provide information about the distances between interacting molecules. By examining which crosslinkers effectively conjugate to particular domains of a protein, information may be obtained about conformational changes that hindered or exposed amino acids in the tertiary and quaternary structure. The use of crosslinkers has made the study of surface receptors much easier. By derivatizing a receptor with a crosslinker before or after contact with the ligand, it is possible to isolate the receptor-ligand complex. The use of radioiodinatable crosslinkers makes it possible to identify a particular receptor by autoradiographic detection.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

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Introduction
How to choose a crosslinker Crosslinkers are selected on the basis of their chemical reactivities (i.e., specificity for particular functional groups) and compatibility of the reaction with the application. The best crosslinker to use for a specific application must be determined empirically. Crosslinkers are chosen based on the following characteristics: • Chemical specificity • Spacer arm length • Water solubility and cell membrane permeability • Same (homobifunctional) or different (heterobifunctional) reactive groups • Spontaneously reactive or photoreactive groups • Cleavability • Reagent contains moieties that can be radiolabeled or tagged with another label Crosslinkers contain at least two reactive groups. Functional groups that can be targeted for crosslinking include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids (Table 1). Coupling also can be nonselective using a photoreactive phenyl azide crosslinker. Our web site (www.thermo.com/pierce) contains a crosslinker selection guide by which the above-listed parameters may be chosen and a list of available crosslinkers with those features generated.
Table 1. Reactive crosslinker groups and their functional group targets.
Reactive Group Aryl Azide Carbodiimide Carbonyl Diazirine Hydrazide Hydroxymethyl Phosphine Imidoester Isocyanate Maleimide NHS-ester PFP-ester Psoralen Pyridyl Disulfide Vinyl Sulfone Target Functional Group Nonselective (or primary amine) Amine/Carboxyl Hydrazine Nonselective Carbohydrate (oxidized) Amine Amine Hydroxyl (non-aqueous) Sulfhydryl Amine Amine Thymine (photoreactive intercalator) Sulfhydryl Sulfhydryl, amine, hydroxyl

intermolecular crosslinking is favored with a crosslinker containing a long spacer arm. Often crosslinkers that are cleavable, non-cleavable and have various spacer arm lengths are used to obtain a complete analysis of protein structure. General reaction conditions In many applications, it is necessary to maintain the native structure of the protein complex, so crosslinking is most often performed using mild pH and buffer conditions. Furthermore, optimal crosslinker-to-protein molar ratios for reactions must be determined. Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low- to moderatedegree of conjugation may be optimal so that biological activity of the protein is retained. The number of functional groups on the protein’s surface is also important to consider. If there are numerous target groups, a lower crosslinker-to-protein ratio can be used. For a limited number of potential targets, a higher crosslinker-to-protein ratio may be required. Furthermore, the number of components should be kept to a minimum because conjugates consisting of more than two components are difficult to analyze and provide less information on spatial arrangements of protein subunits. Water solubility and membrane permeability Many crosslinkers, by virtue of their hydrophobic spacer arms, have limited solubility in aqueous solutions. These crosslinkers are generally dissolved in DMF or DMSO, then added to the biological system or solution of biomolecules to be crosslinked. Hydrophobic crosslinkers are able to cross cellular and organellar membranes and affect crosslinking both at the outer surface of a membrane and within the membrane-bounded space. It is often inconvenient or undesirable to introduce organic solvents into a crosslinking procedure for a biological system. It is also desirable in many instances to effect crosslinking only on the outer surface of a cellular or organellar membrane without altering the interior of the cell or organelle and, in such cases, several water-soluble, membrane-impermeable crosslinkers are available. Some crosslinkers contain a spacer arm formed from polyethylene glycol (PEG) subunits and resulting in a polyethylene oxide (PEO) chain with abundant oxygen atoms to provide water solubility. These crosslinkers are designated by a (PEG)n in their name and are both water-soluble and unable to penetrate biological membranes. They provide the added benefit of transferring their hydrophilic spacer to the crosslinked complex, thus decreasing the potential for aggregation and precipitation of the complex. Other crosslinkers obtain their water-solubility and membrane-impermeability by virtue of a charged reactive group at either end of the spacer. These charged reactive groups, such as sulfo-NHS esters or imidoesters, impart water-solubility to the crosslinking reagent, but not to the crosslinked complex because the reactive group is not a part of the final complex.

Often different spacer arm lengths are required because steric effects dictate the distance between potential reaction sites for crosslinking. For protein:protein interaction studies, the proximity between reactive groups is difficult to predict. Usually, a crosslinker with a short (4-8 Å) spacer arm is used first and the degree of crosslinking determined. A crosslinker with a longer spacer arm may then be used to optimize crosslinking efficiency. Short spacer arms are often used in intramolecular crosslinking studies, and

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For more information, or to download product instructions, visit www.thermo.com/pierce

reaction time and quantity of crosslinker are less critical when using sulfo-NHS-esters. but are not interacting.Crosslinking Applications Crosslinking applications Cell surface crosslinking Crosslinkers are often used to identify surface receptors or their ligands. Some proteins are difficult to study because they exist in different conformations with varying pH or salt conditions. the crosslinking is not dependent on amino acid composition for successful conjugation. intermolecular crosslinking can occur. they are still able to penetrate membranes. carboxyl.or sulfhydryl-reactive reagents are used for identification of particular amino acids or for determination of the number. Dilute protein solutions and high concentrations of crosslinker favor intramolecular crosslinking when homobifunctional crosslinkers are used. but they may result in intermolecular coupling. cleavable crosslinkers with increasing spacer arm lengths may be used to determine the distance between subunits. 21667). the proteins are subjected to two-dimensional electrophoresis. One way to avoid conformational changes is to crosslink subunits. a homobifunctional photoactivatable phenyl azide. Failure to obtain crosslinking with a panel of shorter crosslinkers. protein subunit structure and arrangement. call 800-874-3723 or 815-968-0747.to medium-spacer arm crosslinkers are selected when intramolecular crosslinking is desired. Homobifunctional sulfo-NHS-esters. Upon conjugation the cells may be washed. Surface membrane proteins can conjugate in the presence of water-soluble and waterinsoluble crosslinkers. Subunit crosslinking and protein structural studies Crosslinkers can be used to study the structure and composition of proteins in samples. heterobifunctional sulfo-NHS-esters and photoreactive phenyl azides are good choices for crosslinking proteins on the cell surface. while conjugated subunits will separate according to the combined size. Sulfhydrylreactive crosslinkers may be useful for targeting molecules with cysteines to other molecules within the membrane. with differing spacer arm lengths. For determination or confirmation of the three-dimensional structure. Determination of whether a particular protein is located on the surface or the integral part of the membrane can be achieved by performing a conjugation reaction of a cell membrane preparation to a known protein or radioactive label using a water-soluble or water-insoluble crosslinker. Amine-. To order. It is cleavable and can be radiolabeled with 125 I using Thermo Scientific Pierce Iodination Beads (Product # 28665). Therefore. can crosslink between subunits without crosslinking to extraneous molecules if used in optimal concentrations and conditions. Membrane-impermeable crosslinkers ensure cell-surfacespecific crosslinking. In the first dimension. Homobifunctional NHS-esters. enzyme:substrate interactions. such as aminereactive Thermo Scientific Pierce DFDNB (Product # 21525) or the photoactivatable amine-reactive crosslinker Thermo Scientific Pierce NHS-ASA (Product # 27714). but the cleaved subunits will be off the diagonal. lipids or other molecules inside and outside the membrane. can be used to crosslink proteins to associated molecules within the membrane to determine the distance between molecules. contact your local branch office or distributor. Thermo Scientific Pierce BASED (Product # 21564). 3 . both of the dissociated molecules will still be iodinated. The hydrophilic character of EDC can result in much different crosslinking patterns in membrane and subunit studies than with hydrophobic carbodiimides such as DCC. The molecular weights of the individual subunits should be compared with predetermined molecular weights of the protein subunits using reducing SDS-polyacrylamide gel electrophoresis. Some subunits may not be crosslinked and will separate according to their individual molecular weights. imidates or heterobifunctional NHSester/photoactivatable phenyl azides are commonly used for these procedures. while obtaining conjugation with the use of longer reagents. Because both reactive groups on this crosslinker are nonspecific. Cell membrane structural studies Cell membrane structural studies require reagents of varying hydrophobicity to determine the location and the environment within a cell’s lipid bilayer. waterinsoluble dicyclohexylcarbodiimide (Thermo Scientific Pierce DCC. Slightly longer crosslinkers. generally indicates that the molecules are located in the same part of the membrane. Successful crosslinking with shorter crosslinkers is a strong indication that two molecules are interacting in some manner. The sulfonyl groups attached to the succinimidyl rings of NHS-esters result in a crosslinker that is water-soluble. 22981). can also crosslink between subunits. Water-insoluble crosslinkers when used at controlled amounts of reagent and reaction time can reduce membrane penetration and reaction with inner membrane proteins. but not in the presence of water-soluble crosslinkers. the proteins are separated using non-reducing conditions and the molecular weights are recorded. Fluorescent tags are used to locate proteins. After cleavage. Carbodiimides that result in no spacer arm. solubilized and characterized by SDS-polyacrylamide gel electrophoresis (PAGE) to determine whether the protein of interest was conjugated. Once conjugated. Outside the United States. Crosslinked subunits that were not reduced will produce a diagonal pattern. Product # 20320) and other water-soluble/-insoluble coupling reagent pairs are used to study membranes and cellular structure. Experiments using crosslinkers with different reactive groups may indicate the locations of specific amino acids. location and size of subunits. Although imidoester crosslinkers (imidates) are watersoluble. such as Thermo Scientific Pierce DMP (Product # 21666. along with short-length conjugating reagents. Thermo Scientific Pierce EDC (Product # 22980. is one of the more versatile crosslinkers for the study of protein interactions and associations. Adjusting the reagent amount and protein concentration can control intermolecular crosslinking. Often it is best to attempt crosslinking with a water-soluble and water-insoluble carbodiimide to obtain a complete picture of the spatial arrangements or protein:protein interactions involved. and cell-surface and membrane receptors. Short. The second dimension of the gel is then performed using conditions to cleave the crosslinked subunits. Various crosslinkers. If the spacer arm is too long. Integral membrane proteins will form a conjugate in the presence of a water-insoluble crosslinker. The individual molecular weights of the crosslinked subunits can be determined. membrane-impermeable and nonreactive with inner-membrane proteins.

then reduced to yield sulfhydryls. 10.thermo. 22981 22360 22322 22311 22312 22416 22317 22309 22324 4 For more information. For several years. pyridyl disulfide crosslinker used to conjugate aminecontaining molecules to sulfhydryls. Because the distances between two molecules are not always known. This method can be very efficient and yield an immunogen that is capable of eliciting a good response upon injection. releasing the toxin within the targeted cell. Carbodiimides are good choices for producing peptide-carrier protein conjugates because both proteins and peptides usually contain several carboxyls and primary amines. Thermo Scientific Pierce Crosslinkers commonly used to produce immunogens. Occasionally. or to download product instructions. et al. refer to the free Antibody Technical Handbook (Product # 1601672). amine-reactive NHS-esters or imidates and heterobifunctional. For more information on preparation of immunogen conjugates. Thermo Scientific Pierce SPDP (Product # 21857) is a reversible NHS-ester. sulfhydryls must be introduced into them by reduction of disulfides.com/pierce . the conjugate must be stable in vivo. this has been the “workhorse” crosslinker for production of immunotoxins. Traut’s Reagent reacts with amines and yields a sulfhydryl when its ring structure opens during the reaction. The amine-reactive NHS-ester is usually reacted with the antibody first. visit www. Table 2. Crosslinker EDC SMCC Sulfo-SMCC MBS Sulfo-MBS SMPB Sulfo-SMPB GMBS Sulfo-GMBS Reactivity Amine/Carboxyl Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Product # 22980. Creation of immunotoxins Specific antibodies can be covalently linked to toxic molecules and then used to target antigens on cells. phenyl azides are very useful for this type of crosslinking because they usually result in efficient crosslinking.. (2001). Homobifunctional. Often peptides are synthesized with terminal cysteines to enable attachment to supports or to carrier proteins using sulfhydryl-/amine-reactive. or through chemical modification reagents. K. which is common for procedures involving ricin A chain and abrin A chain. they proceed to kill the cell by ribosome inactivation or other means.Crosslinking Applications Protein interactions and associations Crosslinkers are used for identification of near-neighbor protein relationships and ligand-receptor interactions. amine-reactive. Thermo Scientific Pierce SASD (Product # 27716) is a unique sulfo-NHS-ester. For immunotoxins to be effective. Another chemical modification reagent that is commonly used for production of immunotoxins is 2-iminothiolane. also known as Traut’s Reagent (Product # 26101). A second SPDP molecule can be used for this purpose and is reacted with amines on the immunotoxin.N. toxins do not contain surface sulfhydryls. a sulfhydryland amine-reactive crosslinker. 1293-1304. the optimal length of the spacer arm of the crosslinker may be determined by the use of a panel of similar crosslinkers with different lengths. disulfide-containing conjugates have been shown to be more cytotoxic to tumor cells than noncleavable conjugates of ricin A immunotoxins. therefore. For more information on this type of application for crosslinkers. photoactivatable phenyl azides are the most commonly used crosslinkers for these applications. Cells are able to break the disulfide bond in the crosslinker. Quantitative evaluation of the length of homobifunctional protein crosslinking reagents used as molecular rulers. In addition. Carbodiimides such as EDC react with carboxyls first to yield highly reactive unstable intermediates that can then couple to primary amines. Immunotoxins are brought into the cell by surface antigens and. The type of crosslinker used to make an immunotoxin can affect its ability to locate and kill the appropriate cells. The crosslinkers chosen for these applications are usually longer than those used for subunit crosslinking. Often these antibodies are specific for tumor-associated antigens. such as Thermo Scientific Pierce Sulfo-SMCC (Product # 22322). The best crosslinker to use depends on the functional groups present on the hapten and the ability of the hapten-carrier conjugate to function successfully as an immunogen after its injection. once the immunotoxin reaches its target. In general. photoactivatable phenyl azide that is both iodinatable and cleavable that allows for detection and analysis of small quantities of protein. Suggested Reading For more information concerning accurate measurements of 32 popular crosslinkers using stochastic dynamics calculations. the creation of immunogens Many crosslinkers are used for making conjugates for use as immunogens (Table 2). heterobifunctional crosslinkers. Both cleavable or noncleavable crosslinkers can be used. Thermo Scientific Pierce DSS (Product # 21555) or its cleavable analog DSP (Product # 22585) are among the shorter crosslinkers used for protein:protein interactions. see the following reference: Houk. once internalized. Carrier protein conjugation. the antibody must be separable from the toxin to allow the toxin to kill the cell. may be used if one of the two proteins or molecules is known to contain sulfhydryls. refer to the free Protein Interaction Technical Handbook (Product # 1601672). Thiolcleavable. Protein Sci. NHS-ester.

Conjugates are often prepared by attachment of an enzyme. Outside the United States. Sulfo-SMCC is first conjugated to one protein. however. Product # 22411).. For more information on solid-phase immobilization. Spacer arms help to overcome steric effects when the ligand is immobilized too near the matrix to allow access by the receptor. the other component is activated with the NHS-ester/aldehyde (e. Product # 22411. In this strategy. disulfides in the protein may be reduced. To order. agarose. Heterobifunctional crosslinkers are perhaps the best choices for antibody-enzyme or other protein:protein crosslinking. Although secondary antibody conjugates are available and relatively inexpensive. For some compounds that are difficult to immobilize.g. Heterobifunctional photoactivatable phenyl azide crosslinkers are seldom used for making protein:protein conjugates because of low conjugation efficiencies. but they often yield conjugates that produce high background in immunoassays. Homobifunctional NHS-ester or imidoester crosslinkers may be used in a one-step protocol but polymerization and selfconjugation are also likely. After the antibody binds to the Fc-binding proteins. Useful spacers are diaminodipropylamine (DADPA).Solid-phase immobilization Proteins. phenyl azides to attach them to amine-activated supports. Homobifunctional sulfhydryl-reactive crosslinkers such as BMH (Product # 22330) and DPDPB (Product # 21702) may be useful if both proteins to be conjugated contain sulfhydryls. Carbodiimides such as DCC (Product # 20320) and EDC (Product # 22980. Glutaraldehyde conjugates are easy to make. Steric effects are usually most pronounced when the ligand is a small molecule. and the second is thiolated with SATA (Product # 26102) or Traut’s Reagent (Product # 26101). These conjugates often result in less background in enzyme immunoassays and are relatively easy to prepare. contact your local branch office or distributor. The hydrazide. Heterobifunctional crosslinkers that can be reacted in two steps are often more useful and efficient for producing solid-phase supports than homobifunctional crosslinkers.. Antibody-enzyme conjugates (primary or secondary antibodies) are among the most common protein:protein conjugates used. The crosslinkers DMP (Product # 21666) and DSS (Product # 21555) are used to immobilize antibodies on Protein A or Protein G supports for antigen purification. Many reagents are used for the production of antibody-enzyme conjugates. The supports may be nitrocellulose or other membrane materials. the antibody is oriented so that the Fab region is available for antigen binding.g. enzyme-labeled primary antibodies are usually expensive and can be difficult to obtain. 22419) chemistry. or if NHS (Product # 24500) or Sulfo-NHS (Product # 24510) is used to enhance the reaction. polystyrene plates or beads. ethylenediamine.and aldehyde-activated components are then mixed together and spontaneously react to form the specific conjugate. Product # 26147) are based on this chemistry. DSS or DMP is applied to the bound antibody column to link the two proteins through primary amines. Carbodiimide procedures are usually one-step methods. Amine-activated supports can be converted to sulfhydryl-reactive supports using NHS-ester maleimide crosslinkers such as Sulfo-SMCC (Product # 22322).. peptides and other molecules can be immobilized onto solid supports for affinity purification of proteins or for sample analysis. Any of the other NHS-ester..and amine-activated glass. it may be possible to use NHS-ester. Product # 22419). 6-amino-caproic acid and any of several amino acids or peptides. Another strategy for creating specific protein conjugates without the risk of self-conjugation takes advantage of a two-step NHS-ester/hydrazide and NHS-ester/aldehyde (e. plastic and agarose supports. 5 . Carbohydrate moieties can be oxidized and then coupled to primary amines on enzymes in a procedure called reductive alkylation or amination. or glass slides. beaded polymers. Some supports can be activated for direct coupling to a ligand. Unwanted self-conjugation inherent when using homobifunctional NHS-ester reagents or glutaraldehyde can be avoided by using a reagent such as SMCC (Product # 22360) or Sulfo-SMCC (Product # 22322). two-step methods are possible if reactions are performed in organic solvents. call 800-874-3723 or 815-968-0747.g. photoactivatable. while in a separate reaction. Protein:protein conjugates One of the most used applications for crosslinkers is the production of protein:protein conjugates. Alternatively. 22981) are very useful for coupling proteins to carboxy. These same reagents are also useful for activating surfaces to which a biomolecule is to be bound. some self-conjugation of the antibody may occur. maleimide or pyridyl disulfide crosslinkers can be substituted for Sulfo-SMCC in this reaction scheme. The photoactivatable phenyl azide becomes reactive once it is exposed to the appropriate wavelength and is able to nonselectively couple to almost any ligand. one component of the conjugate is activated with the NHS-ester/hydrazide (e. however. followed by conjugation. fluorophore or other molecule to a protein that has affinity for one of the components in the biological system being studied. refer to the free Protein Purification Technical Handbook (Product # 1601617).g. EDC is useful for coupling ligands to solid supports and to attach leashes onto affinity supports for subsequent coupling of ligands. Thermo Scientific Pierce Crosslink IP Kit (e. Other supports are made with nucleophiles or other functional groups that can be linked to proteins using crosslinkers. and the two activated proteins are incubated together to form conjugates free of dimers of either protein. hexanediamine.

or to download product instructions. To assist in these crosslinking methods. the fluorescently labeled interacting proteins can be followed in cells to determine the site of interactions or the fate of the proteins after interacting. The sulfhydryl-reactive group of APDP offers the advantage of allowing the course of the bait protein coupling to be monitored by following the loss of the 2-pyridyl-thione moiety (leaving group). Other specialized chemistries are reviewed in Hermanson’s book. They require labeling with 125I before use.or sulfhydryl-reactive crosslinkers can be used for their conjugation to proteins.. visit www. New non-isotopic reagents and methods continue to make this technique more accessible and simple to perform by any researcher. Disadvantages of traditional bifunctional label transfer reagents Although these reagents have been used successfully to obtain data on protein interactions. 1. causing nonspecific labeling of the protein(s) in the mix. DNA probes are often synthesized with primary amines or thiols attached to specific bases. Fluorescent constituents designed into cleavable photoreactive crosslinkers make possible transfer of a fluorescent label to an unknown interacting protein. Label transfer Label transfer involves crosslinking interacting molecules (i. In this case. Thermo Scientific Pierce SFAD: fluorine label transfer reagent SFAD (Product # 27719) is a heterobifunctional crosslinker containing an amine-reactive NHS-ester moiety at one end and a photoreactive perfluorophenyl azide moiety at the other end. amine. They were designed such that the photoreactive moiety bears the transferable label.08 x 103 M-1cm-1). After insertion of the bases into DNA. These trifunctional reagents can be designed to include non-radioisotopic labels such as biotin. The photoactivation step can result in several unproductive pathways that lower crosslinking yield between bait and prey.e. typically through available amine groups. and the efficiency of label incorporation is low. The 125I label can be released during the light reaction. The reagent is non-fluorescent prior to exposure to UV light. More recent offerings have been prepared as trifunctional reagents that more adequately segregate the reactive sites from the label. The first reagents employed using this method were bifunctional. The phenolic hydroxyl activates the ring for substitution reactions to occur ortho or para to its position.com/pierce . The sample is exposed to UV light to photo-crosslink the interacting complex. The user should be aware of the following characteristics of these reagents. EDC has been reportedly used to crosslink RNA to ribosomal protein subunits. but upon photolyzing and coupling to interacting proteins. the crosslinker is first radioiodinated and then reacted with a bait protein. Photoreactive and labeled chemical groups are the same. An example of this type of reagent that incorporates a coumarin group is SAED (Product # 33030). 28666). 3. Thermo Scientific Pierce SAED: fluorescent label transfer reagent Subsequent designs of bifunctional label transfer reagents used nonradioactive labels to avoid the safety issues posed by 125I. bait and prey proteins) with a labeled crosslinking agent and then cleaving the linkage between bait and prey such that the label remains attached to the prey (Figure 1. Bioconjugate Techniques. The reagent also has a disulfide bond that can be reduced. interacting protein. it becomes highly fluorescent. 2.Crosslinking Applications DNA/RNA crosslinking to proteins Crosslinking of DNA or RNA to proteins is more limited because the reactivities of most crosslinkers favor protein:protein crosslinking over protein:DNA crosslinking. photoreactive ring. At this point. APDP contains the sulfhydryl-reactive pyridyl-dithio group. 6 For more information. the label can facilitate detection of the interacting proteins or the complex can be cleaved and the radiolabel transferred to the protein interacting with the bait. 4. page 7). The now radiolabelled. This method allows a label to be transferred from a known protein to an unknown. separated by a cleavable disulfide bond. resulting in cleavage of the crosslinked proteins and transfer of the label to the unknown interacting species. Also the improved stability of the perfluoroaryl nitrene-reactive intermediate provides additional efficiency of insertion into C-H bonds compared to the amine nucleophile reaction typical of non-fluorinated aryl nitrenes that quickly undergo ring expansion. Label transfer is particularly valuable because of its ability to identify proteins that interact weakly or transiently with the protein of interest. The presence of fluorine allows the label transfer process to be monitored by 19F NMR. 2nd Edition (Product # 20036). The 2-pyridyl-thione can be detected at 343 nm (extinction coefficient: 8.thermo. This modified protein is then introduced into a sample and allowed to interact with other proteins. These molecules are either amine-reactive or sulfhydryl-reactive and are labeled radioisotopically with 125I. they possess some inherent deficiencies compared to trifunctional reagents designed for label transfer applications. Iodination of the crosslinker with 125I prior to its use will result in a radioactive label transfer reagent that can tag an unknown interacting protein with a radiolabel after cleavage of the crosslinker’s spacer arm. In practice. The label can then be used to purify and/or detect the interacting protein. which has been substituted with an azido group on the aromatic. Thermo Scientific Pierce APDP: radiolabel transfer reagent APDP (Product # 27720) is a heterobifunctional crosslinker containing a photoreactive group that can be labeled with 125I. Traditional label transfer reagents The earliest examples of label transfer reagents incorporated a photoreactive phenyl azide group that contained a hydroxy-phenyl modification on the ring. unknown protein(s) can be detected by autoradiography after separation by electrophoresis and Western transfer. These compounds can be radioiodinated using typical oxidation reagents such as chloramine T or Thermo Scientific Pierce Iodination Beads (Product # 28665.

1. contact your local branch office or distributor. To order. 3. Transfer proteins to a membrane. Outside the United States. 5. Transfer the Biotin label from the Bait Protein 1 to the Bait Protein 2. Incubate in the dark 30-60 minutes. 4. in the dark.O HN NH Biotin S SO 3 - Cleavable Disulfide S S O || O O N O Amine-reactive Photoreactive O N3 NH HN S S | S O || NH | Protein 1 Protein 2 NH2 Biotin Label Transfer Reagent Protein 1 NH2 O HN NH S O || S S N H Protein 2 N3 NH2 Protein 1 + O || SO 3 - N3 UV UV HO-N O HN NH S S | S O || N–H Figure 1. || HS O Protein 1 Protein 2 N N H DTT O O || N–H | Protein 1 NH HN S SH Protein 2 N N H 7 . Reduce the disulfide bond using DTT. 2. General scheme for label transfer reactions. call 800-874-3723 or 815-968-0747. Introduce Biotinylated Bait Protein to Prey Protein (Protein 2) containing sample under conditions which promote favorable binding. 30 minutes at RT]. Detect Bait or Prey Proteins with the appropriate antibodies or Streptavidin-HRP. Reduced sample is applied to a gel and separated by electrophoresis. Capture the Bait:Prey complex by irradiating the photoreactive aryl azide group with UV light. React available amine groups on a purified Bait Protein (Protein 1) with Sulfo-NHS containing Biotin Label Transfer Reagent [pH 7-9.

59. 186. et al. 273(3). Biochem. (1994).. 208-221. thiol-cleavable label transfer reagents enable the tagging of a prey protein. D. Biophys. 14. A Practical Approach. Anal.. M.. R. Biol. 101.R. et al. 15. 763-766. 265(21). Q. 5880-5886. Chem. Pandurangi. 8 For more information. 5. 50/pkg. p. Em: 440-460 nm • No radiolabeling required • AMCA moiety exhibits large Stokes shift • Improved photoconjugation efficiency • Photolyzes at 320 nm • Label transfer monitored by 19 F NMR • Water-soluble • Cleavable • Amine-reactive 16.15 M.W.W. Chem.W. et al. 65(2).Biol. et al. 264(9). K. Chem.W. Dey. 446. Chem. 59. Photochem.6 Å M. J.48 Spacer Arm 14.3´dithiopropionate Sulfo-SFAD O Na+ O– S O O O N O O M. 621. Photobiol. 1670-1676. A. et al. Sato. Size 50 mg APDP M. visit www.6 Å N N+ N– SFAD Compatible Pierce products for addition of 125I to APDP. Protein Function..10.0 Å 33030 Sulfosuccinimidyl 2-(7azido-4-methylcoumarin3-acetamido)ethyl-1. 2168-2172.A.. 13. Biol. (1989). Biol.W. (1998).55 Spacer Arm 21. A. R..3 dithiopropionate Sulfo-SAED O N O O Na+ –O S O O O S S H N O O O N N+ N– M. (1996). 621. J. 2. Res. 28600 Pierce Iodination Reagent (1. (1997). N. Ghinea. 10. Felin. (1996). 7. (1990).... J. et al. 271(52).G. Chem.G. 6. Thevenin. 201-212. et al. 8. Lam..thermo. W. 4. and Mosher. (1988). Oxford:IRL Press. Pandurangi. J..6 Spacer Arm 23. et al.17 5 mg 9.S. 33284-33292. D. 4755-4758. et al. Chem. et al. (1997). R.W. The photolysis wave lengths for these reagents are in the range between 320-400 nm. Biol. 11.6 Å 27719 Sulfosuccinimidyl(perfluoroazidobenzamido)ethyl-1. Biol. 3. D.com/pierce . J. 358a. 17. (1991).A. Nature 416 (April 19). (1991).4. Kang. (1992).6 Å O S S N H F F F F SAED • Water-soluble • Amine-reactive • Photoreactive • Prey protein tracked by fluorescence • Ex: 345-350 nm. 50 mg 14. 1-5 Pkg. Y. J.. Chem. 9(2).6-Tetrachloro-3α. limiting damage to biomolecules by irradiation. 23-29. 58-60. B. J.F. Ordering Information Product # Description 28601 Pierce Pre-Coated Iodination Tubes (12 mm x 75 mm glass test tubes coated with 50 µg Pierce Iodination Reagent in 100 µl volume) Pkg. 597. Size 10 tubes/ pkg. J. Gao. et al.Crosslinking Applications Label transfer reagents Thermo Scientific Pierce Bifunctional label transfer reagents Heterobifunctional. 265(4). 267. and Frazier. 28665 Pierce Iodination Beads (N-Chloro-benzenesulfonamide modified non-porous polystyrene beads) 28666 Pierce Iodination Beads (N-Chloro-benzenesulfonamide modified non-porous polystyrene beads) 250/pkg. J. Shephard. Ordering Information Product # 27720 Description N-[4-(p-Azido-salicylamido) butyl]-3´-(2´-pyridyldithio) propionamide Structure N S S O H N O N H HO N N+ N– Features • Radioiodinatable between –N3 and –OH group of phenyl ring • –SH-reactive • Reaction monitored at 343 nm • Membrane permeable Ref. 272(9). A.S. Chem. Biophys. Bioorg.W. Smith.3. Biol. or to download product instructions.. Chattopadhyay.48 Spacer Arm 14. Zhang. Glycobiology 7(1). and Cheresh. 29650-29657. (1997).60 Spacer Arm 23. (1989).A. et al. Traut. (2002). J. 306-313. et al. 597. Biol.6α-diphenylglycoluril) 1g References 1. J. 16. Lala. (1990). photoreactive. 12. J. Pept. J. 26(4). 269(47). (1998). 249a. 12323-12329. Smith. 9. 12267-12271. et al. Chem. E..

7 Å 21. Outside the United States. +O – To order. It contains an amine-reactive sulfo-NHS-ester on one arm (built off the α-carboxylate of the lysine core). 879. Exposure to UV light (300-366 nm) couples the photo-reactive end to the nearest available C-H or N-H bond in the bait:prey complex. a bait protein first is derivatized with Sulfo-SBED through its amine groups. biotin can operate as a handle for purification of the prey protein or prey protein fragments or as a detection target using streptavidin-HRP and colorimetric or chemiluminescent substrates. The biotin label can be used to significant advantage in a label transfer application. with a biotin label designed into Sulfo-SBED.98 Spacer Arms Phenyl azide Biotin Biotin 14. which permits transfer of the biotin component to any captured proteins. In use. call 800-874-3723 or 815-968-0747.Sulfo-SBED Reagent Thermo Scientific Pierce Sulfo-SBED: Label transfer protein interaction reagent Label transfer reagents can also have biotin built into their structure. radiolabeling with 125I is no longer necessary. Thermo Scientific NeutrAvidin Protein or monomeric avidin. N– N+ N O HN Photo-reactive 21. facilitating isolation or identification of the unknown species using streptavidin.2 Å Amine-Reactive S Na Figure 2. The architecture of this trifunctional label transfer reagent differs substantially from the bifunctional counterparts discussed above. and the modified protein is allowed to interact with a sample.7 Å S S Cleavable Disulfide O O N O O O O NHS ester NHS ester Phenyl azide Sulfo-SBED M. the biotin handle remains attached to the protein(s) that interacted with the bait protein.3 Å HN O 24. This type of design allows the transfer of a biotin tag to an interacting protein after cleavage of a cross-bridge. Most importantly. Structure of Sulfo-SBED. The arm containing the sulfo-NHS-ester has a cleavable disulfide bond. For example. The reactive moieties are well-segregated within Sulfo-SBED. a photoreactive phenyl azide group on the other side (synthesized from the α-amine) and a biotin handle (connected to the ε-amino group of lysine). Sulfo-SBED (Product # 33033) is an example of such a trifunctional reagent (Figure 2). The advantages become almost immediately apparent just by examining the structure.2 Å H N O O HN NH Biotin S 14.3 Å 24. Upon reduction and cleavage of the disulfide spacer arm. contact your local branch office or distributor. 9 . resulting in covalent crosslinks between bait and prey.W.

122. Soc. Biochemistry 39.. Chem. the number of literature references for use of Sulfo-SBED in protein interaction-related applications has grown rapidly. et al. Chem. R850-857. (2000). et al. The biotin-labeled prey protein or prey protein peptides recovered as result of the strategies outlined below can be subjected to several detection and identification options designed to discover the identity of the prey protein.A.1 mg 8 x 7. et al. 4. Neely. et al. Ishmael. Chem. K. 7.. Science 279(5349). Biol. S.com/pierce .. R. 5. 10K MWCO 8 x 1 mg 1 pack 200 ml 0. 3. Life Sciences 56 (11/12). 8. Sharma. or to download product instructions. J. Ordering Information Product # Description 33033 33034 33073 Sulfo SBED Biotin Label Transfer Reagent Sulfo-SBED Biotin Label Transfer Reagent. 6. J. 20555-20562. (2002).D.thermo. (2002). No-Weigh Format BupH™ Phosphate Buffered Saline Label Transfer Buffer (20X) Streptavidin-Horseradish Peroxidase Conjugate Dithiothreitol (DTT). 10. (2000). 11. BioConjuate Chem. et al. Horney.C. et al.. Curr. et al. (2000). 9..Crosslinking Applications Applications for Sulfo-SBED Since the first availability of this patented reagent in 1994. No-Weigh Format Slide-A-Lyzer® MINI Dialysis Units Plus Float. et al. (2000). Includes: Sulfo-SBED. Alley. (2001). 9055-9061.K. 2880-2889.E. Daum. Biol. Minami. 3767-3771. In the trypsin digestion strategy. (1998). (1995).. J. 277(23). 6126-6127. L. Am. 1615-1625. Nature Cell Biology 3. D. (1995). 22(6). (2000). 1092-1100. Chem. Cell.J. Biol. Biology 10(23).C. Y. et al. Jacobson. Size 10 mg 8 x 1 mg Kit Sufficient reagents for 8 label transfer reactions for subsequent Western blot analysis. 373-377. J.T. 6. No-Weigh™ Format Sulfo-SBED Biotin Label Transfer Kit Western Blot Application Pkg. et al. Ilver. 12. K. R. 275(12). F. (2001). Trotman. S1-S2. Kleene..7 mg 10 units/pack References 1. Biol. 275(6). J. 502-506 10 For more information. K. Published applications show how Sulfo-SBED can used to: • Define interactions of complexes with activator domains1 • Clarify the mechanism of protein complex assembly2 • Convert to a sulfhydryl-reactive trifunctional reagent to map interactions3 • Study docking site and factor requirements for binding4 • Describe binding contacts of interactors5 • Confirm recognition of a specific phosphoepitope6 • Search for putative binding partners7 • Gain insight into chaperone-mediated refolding interactions8 • Investigate mechanism of protein interaction9 • Facilitate receptor activity-directed affinity tagging (re-tagging)10 • Detect low-abundance protein receptors • Find protein:carbohydrate interactions • Understand drug-receptor interactions11 • Quantitate triple helix-forming oligonucleotides12 Routes to determining the prey protein identification using Sulfo-SBED are outlined schematically in Figure 3. the peptide(s) trapped can offer information relating to the binding interaction interface. M... 823-830. Mol. Chem. J..R. Biol. and Neumann. 276(4).. Geselowitz. D. et al. 9893-9900.A. Note that the biotin label is a purification handle for captured prey protein. 2. visit www.

Outside the United States. contact your local branch office or distributor.Figure 3. Applications of Sulfo-SBED in protein interaction studies. call 800-874-3723 or 815-968-0747. 11 .2 B Protein 1 — S-S ––––––– N3 + Protein 2 UV 300-366 mm 5-15 minutes B Protein 1 —– S-S ———— Protein 2 Reduce Digest with Trypsin B Protein 1 — SH + HS ——— Protein 2 SDS-PAGE B —– S-S ———— Reduce B Protein 1 Protein 2 sA Western Transfer Isolate biotin-containing fragment over immobilized streptavidin or monomeric avidin SH sA — SH + HS ——– B Elute and separate via reversed-phase HPLC Detect with: Streptavidin-HRP or Anti-Biotin Ab or Ab against Protein 2 with HRP-labeled secondary Ab. Applications of Sulfo-SBED in Protein Interaction Studies B Protein 1 + sNHS – S-S ––––––– N3 Dark reaction pH 7. SH B Western Blot Detection Sequence Analysis Mass Spec Identification Legend: sNHS Sulfo N -Hydroxy succinmide ester S-S Disulfide bond N3 Phenyl azide B Biotin To order.

95 Spacer Arms Mts → Atf 11.3 Å Atf → Biotin 30.2 Å O F NH F F Mts-Atf-LC-Biotin C38H56F4N10O8S3 M.3 Å HN O HN O N H O F H N O O NH HN S 30. When exposed to UV-light.3.thermo. Purified bait protein is labeled at reduced cysteine residues.3 Å Atf → Biotin 30. or to download product instructions. 953. allowing one to more precisely explore interaction distances 11. If desired.7 Å F +N N –N Ordering Information Product # Description 33093 Mts-Atf Biotin Label Transfer Reagent 2-[N2-(4-Azido-2.com/pierce .1 Å F F –N N+ CH3 O O S S 29.7 Å N F Mts-Atf-Biotin C32H45F4N9O7S3 M. rapid and quantitative labeling of the bait protein • Tetrafluorophenyl azide moiety reacts three. increasing the likelihood of capturing sufficient bait:prey complex to detect • Sulfinic acid byproducts of the Mts reaction do not interfere with disulfide bond formation or the activity of the bait protein and decomposes quickly to a volatile low molecular weight product • Disulfide bond spacer arm connecting bait and prey proteins is easily reversed with commonly used reducing agents DTT. Reducing the disulfide-bond releases the bait protein and leaves the biotin label on the prey. Reaction of Mts-Atf-Biotin with bait protein containing sulfhydryls (reduced disulfide bonds). Size 5 mg Product # Description 33083 Mts-Atf-LC Biotin Label Transfer Reagent 2-[N2-[N 6-(4-Azido-2.8 Å HN S 35. Highlights: • Mts moiety is highly specific for the sulfhydryl (–SH) group that occurs in the side chain of reduced cysteine residues. 12 For more information.1 Å Mts → Biotin 29.11 Spacer Arms Mts → Atf 11.W. then allowed to form an interaction complex with the prey protein. Size 5 mg CH3 O O S S SH SH HN O Biotin Arm O F –N HO S O N+ N F HN O Biotin Arm + F –N N+ F HN F HN O F N F F Bait protein containing sulfhydryls Mts-Atf-Biotin Methylsulfinic acid Activated Bait Protein Figure 4. the activated bait protein may be allowed to interact with other proteins (the prey) and then crosslinked together by UV-activiation of the tetrafluorophenyl azide group. enabling precise. 2-mercaptoethanol or TCEP • Mts reaction and photoreaction are compatible with physiologic buffer conditions required for most protein interactions • Long chain (LC) and short chain versions are offered.3. 839.7 Å CH3 O O S S O HN O N H O H N O NH 29. Once desalted to remove excess nonreacted Mts-Atf-Biotin and byproducts (methylsulfinic acid).Crosslinking Applications Thermo Scientific Pierce Mts-Atf-Biotin Label Transfer Reagents Sulfhydryl-directed.W.5. the disulfide bond in the Mts-Atf-Biotin may be cleaved with a reducing agent.3 Å HN O 21. the photoreactive group activates to form covalent bonds to adjacent sites on the prey protein. photoreactive biotin label transfer reagents. These two Pierce Biotin-containing Reagents incorporate the benefits of the sulfhydryl-specific methanethiosulfonate (Mts) group and the high-yielding photoreactive tetrafluorophenylazide moiety.6-tetrafluorobenzoyl6-aminocaproyl)-N 6-(6-biotinamidocaproyl)-Llysinylamido)]ethylmethanethiosulfonate SH S S Pkg. powerful new reagents for protein interaction analysis were created.to four-times more efficiently than regular phenyl azide moieties.6-tetrafluorobenzoyl)-N 6(6-biotinamidocaproyl)-L-lysinyl] ethylmethanethio-sulfonate Ordering Information Pkg. By combining these reactive groups with a biotin tag.1 Å Mts → Biotin 29.5. transferring the biotin label to the prey protein. visit www.

2. Na+O– O S O O N O O O D D BS3-d4 M. 69-76. Back. pp.W.. These reagents react efficiently with primary amine groups (–NH2) at pH 7-9 to form stable amide bonds. Am.1-5 Heavy/light crosslinker pairs Thermo Scientific Pierce BS2G and BS3 are water-soluble.4 Å spacer arm that can act as molecular rulers for estimation of spatial relationships in protein structure-function studies. 16. 13 . Biochem.C. Hermanson. Sci. B..T. 3. (2003). d4 vs d0) facilitates identification of low-abundance crosslinked peptides. BS2G-d4 and BS3-d4 react identically to their H-substituted counterparts. J. Natl. thus conducting linking and labeling in one step. Proc.7 Å O S O O–Na+ O O N O O BS2G-d0 M. call 800-874-3723 or 815-968-0747. The strategy requires the availability of both “light” or hydrogen-containing and “heavy” or discretely substituted deuterium analogs of crosslinking agents.2. 1225-1237.7-suberate-d4 BS2-G-d0 BS2-G-d4 Bis(Sulfosuccinimidyl) glutarate-d0 Bis(Sulfosuccinimidyl) 2. Ordering Information Product # Description 21590 21595 21610 21615 BS -d0 3 Pkg. Heavy and light analogs are reacted simultaneously with the target protein or protein complex. Mass Spectrom. Anal. S. et al. et al.. The results positively identify the crosslinked peptides. Acad. Bioconjugate Techniques. Further analysis of the reaction products resulting from the simultaneous reaction of these heavy and light crosslinkers with a target protein or protein complex is accomplished by MALDI-TOF-MS.Structure determination with heavy/light crosslinker pairs Recently. 154. et al. Krieg. Distance constraints provided by these data can yield low-resolution three-dimensional structure information that can be used to create structural models of the protein in solution. 149-159. ESI-LC/MS/MS or ESI-FTICR-MS. (1986). 530.4. Rapid Commun. A. et al. Use of heavy and light crosslinkers in this application dramatically simplifies identification of the peptides resulting from the coupling reactions.45 Spacer Arm 11. homobifunctional sulfonated N-hydroxysuccinimide esters (Sulfo-NHS esters) with a 7. et al. USA 83. 303-313. Eur.W. Soc.. 2005-2014. The reagents described here are the deuterated and non-deuterated analogs of BS2G and BS3. 14. Trends Biochem. 834-850. Schilling. 572. J. 38. San Diego: Academic Press. A. G. Application of a 1:1 ratio of two identical crosslinking agents differing only in the number of deuterium atoms in their chemical composition (e. (2002).T. 17. Sinz. Muller.R. Protein Function. 687-689. (1996).R. R. 331. 2.4 Å O O O O O N S O O–Na+ O S O O–Na+ O O N O O D D D D O O N O Na+O– S O O O BS2G-d4 M. identification of the large number of crosslinking sites from the complex mixtures generated by chemical crosslinking remains a challenging task. (2005). 416. and Sinz. pp. p. et al. 284. Mass Spectrom. Biol. (2003). J. 77.H. Chem. (1989). differing by four mass units after enzymatic digestion of the crosslinked protein or protein complex. Intermolecular crosslinking of an interacting protein complex and subsequent MS analysis have been successfully applied to determine the contact surfaces of binding partners in a protein complex. 214.W. G. 26.7. K. These reagents are supplied as a sodium salt and are soluble in water at a concentration up to 10 mM.g. Chem.4 Å D D O O O N O O S O O–Na+ Na+O– O S O O N O O O BS3-d0 M. By incorporating an isotopic label into the crosslinking reagent. G. 534.4-glutarate-d4 References 1. Dihazi. Sgro. et al.. 73. has emerged as a strategy to obtain lowresolution three-dimensional structural data of protein structures and protein interfaces in complexes from low quantities of proteins within a relatively short time. S. et al. (2003). However.7 Å Na+O– O O O N O S O O To order. A Practical Approach.Y. Outside the United States.. J. 416. 4. San Diego: Academic Press. Hermanson.. Size 10 mg 10 mg 10 mg 10 mg Bis(Sulfosuccinimidyl) suberate-d0 BS3-d4 Bis(Sulfosuccinimidyl) 2. Anal. Sci. (1996). U. Mass Spectrom. identifies the crosslinked products. 1927-1934 Pearson. J. combined with high-resolution mass spectrometry. 495-503. (2003).35 Spacer Arm 7. Mass Spectrom. D. Mol. 8604-8608. contact your local branch office or distributor. (2001). Rapid Commun. J. chemical crosslinking.W. Peri.7 Å and 11..38 Spacer Arm 7. et al. Proteins generally contain several primary amines in the form of lysine residue side chains and the N-terminus of each polypeptide that are available as targets for the NHS ester-reactive group. 101.. (2001).43 Spacer Arm 11.W. (1986). 5.. Kalkhof. Bioconjugate Techniques. Oxford: IRL Press. M. the crosslinked peptides are identified easily in the presence of the numerous unmodified tryptic peptides. Traut. Isotopic MS patterns. 576.

intramolecular crosslinking and/or polymerization. Heterobifunctional crosslinkers possess two different reactive groups that allow for sequential (two-stage) conjugations. helping to minimize undesirable polymerization or self-conjugation.and heterobifunctional crosslinkers Crosslinkers can be either homobifunctional or heterobifunctional. pyridyl disulfides and α-haloacetyls. Heterobifunctional reagents can be used when modification of amines is problematic. However. carboxyls. Homo. Crosslinkers that are amine-reactive at one end and sulfhydryl-reactive at the other end are especially useful in this regard. Even when conjugation of two different proteins is the goal.. This reactivity allows for specific attachment of the labile thermoreactive group first. EDC.g. 14 For more information. phenols and carbohydrates may be more appropriate targets. the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs. and yields of 10% are considered acceptable. to each other or to stabilize quaternary structure. in solution. These have distinct advantages in protein:protein interaction studies and in cases in which the availability of thermoreactive targetable functional groups is unknown. visit www. The reactivity of the photochemical reagent allows for formation of a conjugate that may not be possible with a group-specific reagent. 22981) and effect direct coupling between carboxylates (–COOH) and primary amines (–NH2) and have been used in peptide synthesis. one-step crosslinking with homobifunctional reagents often results in self-conjugation. NHS-esters react with amines to form amide bonds. Multi-step Reactions In sequential procedures. After removing excess unreacted crosslinker. The sulfhydryl-reactive groups are usually maleimides. The most widely used heterobifunctional crosslinkers are those having an amine-reactive succinimidyl ester (e.Single-step vs.com/pierce . Other moieties such as sulfhydryls.thermo.g. The efficiency of most photoreactive crosslinkers is low. hapten-carrier protein conjugation. heterobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. Homobifunctional crosslinkers have two identical reactive groups and often are used in one-step reaction procedures to crosslink proteins.. Amines are sometimes present at the active sites of proteins and modification of these may lead to activity loss. or to download product instructions. NHS-ester) at one end and a sulfhydryl-reactive group on the other end. subsequently. The NHS-ester reactivity is less stable in aqueous solution and is usually reacted first in sequential crosslinking procedures. SFAD (Product # 27719) is a photoactivatable reagent that contains a perfluorophenyl azide with an insertion efficiency of 70%. Product # 22980. Other heterobifunctional reagents have one reactive group that is photoreactive rather than thermoreactive. A two-step strategy allows a protein that can tolerate the modification of its amines to be coupled to a protein or other molecule having different accessible groups. subunit studies and protein:protein conjugation. Carbodiimides are zero-length crosslinkers (e. conjugation to any adjacent N-H or C-H sites may be initiated through the photoreactive group by activation with UV light.

The hydrazine/carbonyl reaction. molecules or surfaces modified with either hydrazine/ hydrazide or aldehyde moieties have extended stabilities in aqueous environments. 15 .g.and aldehyde-reactive moieties provides a small molecule conjugation chemistry that is easy to use and applicable to almost any conjugation scheme. sodium cyanoborohydride) required • Long-term stability • Biomolecules and surfaces modified with hydrazine/hydrazide or aldehyde groups can be prepared.4) or organic solvents with high efficiency • Hydrazide (SHTH)-reactive moieties are stable indefinitely • Highly efficient coupling chemistry • Reactive moieties do not lead to nonspecific interactions • Conjugation does not result in inter-subunit crosslinking Ordering Information Product # Description 22400 SANH (Succinimidyl 4-hydrazinonicotinate acetone hydrazone) Application: used to convert primary amines to hydrazinopyridine moieties Pkg. The leaving group in the reaction is water and no reducing agents (e. stored and used when needed • Hydrazine (SANH)-reactive moieties are stable for several months • Hydrazide (SHTH)-reactive moieties are stable indefinitely • Can be applied to almost any conjugation scheme • Easy to use • Simple mixing of a hydrazine-/hydrazide-modified biomolecule with an aldehyde-modified biomolecule yields the desired conjugate • Reaction is performed in aqueous buffered solutions (pH 4. Size 10 mg 22405 C6-SANH (C6-succinimidyl 4-hydrazinonicotinate acetone hydrazone) Application: SANH with extended six carbon linker (Succinimidyl 4-hydrazidoterephthalate hydrocholoride) Application: used to convert primary amines to aromatic hydrazide moieties (Succinimidyl 4-hydrazinonicotinate acetone hydrazone) Application: used to convert primary amines to hydrazinopyridine moieties 10 mg 22411 SHTH 10 mg 22419 SFB 25 mg 22423 (C6-Succinimidyl 4-formylbenzoate) Application: SFB with extended six carbon linker C6-SFB 25 mg “Hydrazine” Aldehyde Hydrazone Figure 5. see Appendix I. Simple mixing of a hydrazine-/ hydrazide-modified biomolecule with an aldehyde-modified biomolecule yields the desired conjugate. To order. Highlights: • No hazardous reducing agents (e. sodium cyanoborohydride) are required to stabilize the bond.Bioconjugate Toolkit The next generation of biomolecule immobilization/conjugation.5-7..g. Outside the United States. These groups can be incorporated on any surface and remain active without special handling requirements. The modification of biomolecules or surfaces with hydrazine/ hydrazide. To view structures of these products. call 800-874-3723 or 815-968-0747. contact your local branch office or distributor. Unlike other small molecule conjugation methods such as maleimido/thiol chemistry..

(dextran) 40 kDa 16 For more information. 16-17. 6. M. 132. J. Duncan. (1972). Biochemistry of the SH Group. Jocelyn.W. Methods Enzymol. 20 mg 2 mg Activated Dextran Coupling Kit Couple amine-containing ligands and biomolecules.. 91(8). Appl. Anal. Hermanson.T.. Bioconjugate Techniques. (1982). P. al. Appl.W. Academic Press: London. et. 4. Biochem. J. Biochem. 56-63. al. et.A.Single-step vs. Hashida. Reference Mallia. 40-42. 41-57. R. (1983). A.J. al. 5. or to download product instructions. Highlights: • Reliable noncleavable heterobifunctional crosslinking agent offers proven chemistry. Previews 1(4). Academic Press: London. 2. 4. R. (1984). Ordering Information Product # Description 20890 Aldehyde-Activated Dextran Coupling Kit Includes: Aldehyde-Activated Dextran* [CHO Loading: ~300 moles mole of dextran Sodium Cyanoborohydride BupH Phosphate Buffered Saline Pkg. (1983).thermo. Biochem. Ordering Information Product # Description 23456 Controlled Protein-Protein Crosslinking Kit Includes: Sulfo-SMCC Crosslinking Agent 10X Activation/Conjugation Buffer BupH Phosphate Buffered Saline 2-Mercaptoethylamine•HCI Immobilized Reductant SATA Dimethylformamide (DMF) Hydroxylamine•HCI Dextran Desalting Column Column Extender Cysteine•HCI Ellman’s Reagent Pkg. 2nd Ed. Riddle. (1998). 6.. 68-73. Multi-step Reactions Controlled Protein-Protein Crosslinking Kit Contains everything you need to crosslink two proteins and do it successfully.2 ml 2 mg 1 ml 5 mg 2 x 10 ml 2 ea.K. 3. et. (2008). P. Imagawa.. S. Size Kit 5 x 5 mg 190 mg 1 pack *The average molecular weight of dextran used in these preparations is 40 kDa. highly stable intermediates and efficient formation of the target conjugate • Includes disulfide (S-S) reductants and thiolation reagents • Ellman’s Reagent provides the option to monitor reaction sufficiently References 1. al. et. and Vigna.com/pierce . pp. Size Kit 2 mg 20 ml 2 packs 6 mg 0. visit www. Aldehyde-Activated Dextran Average M.C. G.S. 49-60.

large molar excesses of crosslinker (100. although amidine formation is favored between pH 8-10. Glycine also contains a primary amine and may be used in a similar manner. the amidine is formed directly without formation of an intermediate or side product. structure and protein:protein and protein:lipid interactions. The resulting amidine is protonated and.5 is 7. Outside the United States. These crosslinkers have also been used to determine or confirm the number and location of subunits within multi-subunit proteins. A large excess of Tris at neutral. pH 6.to 1. At higher pH. releasing N-hydroxysuccinimide (Figure 7).0 and 0°C in aqueous environments free of primary amines. The NHS-ester reactions are typically performed between pH 7 and 9 and at 4°C to room temperature from 30 minutes to 2 hours. Amine-containing molecule NHS ester compound Amine bond NHS Figure 7.2 x 10 3 M–1 cm–1 at 260 nm at pH 9. The protein concentration should be kept above 10 µM (50-100 µM is optimal) because more dilute protein solutions result in excessive hydrolysis of the crosslinker. Amine-containing molecule Imidoester compound Amidine linkage Figure 6. Extraneous crosslinking that occurs below pH 10 sometimes interferes with interpretation of results when thiol-cleavable diimidoesters are used. Although these crosslinkers are still used in protein subunit studies and solid-phase immobilization. The molar extinction coefficient for the NHS-ester in 50 mM potassium phosphate buffer.N-amidine derivatives. Reaction times at 4°C are increased four-fold from room temperature incubation times to produce similar efficiencies. therefore.to basic-pH may be added at the end of an NHS-ester reaction to quench it. can be used because it contains only tertiary amines. Primary amines are present in the structure of Tris. therefore. NHS-ester crosslinking reactions are most commonly performed in phosphate. resulting in N. An increase in absorbance at this wavelength is caused by the release of NHS. Water-soluble NHS-esters have a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. This half-life decreases to 10 minutes at pH 8. An intermediate N-alkyl imidate forms at the lower pH range and will either crosslink to another amine in the immediate vicinity. HEPES. The molar extinction coefficient of NHS released by hydrolysis and reaction with a nucleophile is 8. They are advantageous when the presence of organic solvents cannot be tolerated. The extent of the NHS-ester hydrolysis in aqueous solutions free of primary amines may be measured at 260 nm. Other buffers may also be used if they do not contain primary amines. or it will convert to an amidine bond.Crosslinker Reactivities Amine-reactive chemistries Imidoesters Imidoester crosslinkers react with primary amines to form amidine bonds. The reaction with the sulfo-NHS-esters is usually performed in 100% To order. NHS-esters are usually used at two. Hydrolysis rate increases with increasing pH and occurs more readily in dilute protein solutions. for example. Hydrolysis of the NHS-ester competes with the primary amine reaction. Imidoesters can penetrate cell membrane and crosslink proteins within the membrane to study membrane composition. Typically.1-10 mM.5 x 10 3 M–1 cm–1 at 260 nm. Imidoester homobifunctional crosslinkers have been used to study protein structure and molecular associations in membranes and to immobilize proteins onto solid-phase supports. therefore. NHS-esters can be grouped into two separate classes with essentially identical reactivity toward primary amines: water-soluble and water-insoluble. N-Hydroxysuccinimide-esters (NHS-esters) NHS-esters yield stable products upon reaction with primary amines with relatively efficient coupling at physiological pH. 17 . In these experiments. the half-life and reactivity with amines increases. contact your local branch office or distributor.0. HEPES or borate buffers at concentrations between 50-200 mM. Accessible α-amine groups present on the N-termini of proteins and ε-amines on lysine residues react with NHS-esters and form amide bonds. which makes it an unacceptable buffer for NHS-ester reactions. the more stable and efficient NHS-ester crosslinkers have steadily replaced them. bicarbonate/carbonate. Imidoester reaction scheme. but have short half-lives. has a positive charge at physiological pH (Figure 6). A covalent amide bond is formed when the NHS-ester crosslinking agent reacts with a primary amine. Imidoester crosslinkers react rapidly with amines at alkaline pH.6 and 4°C. They also have been examined as a substitute for glutaraldehyde for tissue fixation. As the pH becomes more alkaline.000-fold) and low concentrations of protein (1 mg/ml) are used to favor intramolecular over intermolecular crosslinking. Reaction conditions below pH 10 may result in side reactions. Studies using monofunctional alkyl imidates reveal that at pH <10 conjugation can form with just one imidoester functional group. the amidine bonds formed by imidoester crosslinkers are reversible at high pH and. call 800-874-3723 or 815-968-0747. the concentration of the crosslinker can vary from 0.to 50-fold molar excess to protein depending on the concentration of the protein. NHS-ester reaction scheme. crosslinking is more efficient when performed at pH 10 than at pH 8. Studies performed on NHS-ester compounds indicate the half-life of hydrolysis for a homobifunctional NHS-ester is 4-5 hours at pH 7.

20673).3 ensures sulfhydryl selectivity. which has the potential to react with tyrosine. the iodoacetyl group can react with other amino acids. Sulfhydrylcontaining molecule Maleimide compound Thioether bond Figure 8.0. Avoid exposure of iodoacetyl compounds to reducing agents. Table 3. perform iodoacetyl reactions and preparations in the dark. yield cleavable products and allow for sequential coupling.000-fold faster than with amines. it is possible to achieve greater solubility when the reagent is dissolved in organic solvents such as DMSO (Product # 20688). or if a large excess of iodoacetyl group is used. The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between pH 6. however.5 and 7. Hydrolysis of maleimides to a nonreactive maleamic acid can compete with thiol modification. Available NHS-ester haloacetyl crosslinkers are listed in Table 3. then added to the aqueous reaction mixture. To limit free iodine generation.thermo. Active halogen reaction scheme. histidines or methionines. Using a slight excess of the iodoacetyl group over the number of sulfhydryl groups at pH 8. Reagent SIA SIAB Sulfo-SIAB Reactivity Amine/Sulfhydryl Amine/Sulfhydryl Amine/Sulfhydryl Product # 22349 22329 22327 18 For more information. respectively. visit www.com/pierce . sulfhydryls can be introduced into molecules through reaction with primary amines using 2-Iminothiolane or Traut’s Reagent (Product # 26101).5-10% final volume in the aqueous reaction. the reaction favors primary amines.Crosslinker Reactivities aqueous solutions.5 and forms a stable thioether linkage that is not reversible (Figure 8). but the incubation must proceed for longer than one week. maleimides react with sulfhydryls 1. Sulfhydryl-reactive chemistries Maleimides Coupling through sulfhydryl groups is advantageous because it can be site-directed. histidine and tryptophan residues. The solubility of the NHS-esters will vary with buffer composition. The reaction of the iodoacetyl group with a sulfhydryl proceeds by nucleophilic substitution of iodine with a thiol producing a stable thioether linkage (Figure 9). The water-insoluble crosslinkers do not possess a charged group and are lipophilic and membrane-permeable.9-7. Thiols must be excluded from reaction buffers used with Sulfhydrylcontaining molecule Iodoacetyl compound Thioether bond Figure 9. maleimides because they will compete for coupling sites. Haloacetyls The most commonly used α-haloacetyl crosslinkers contain the iodoacetyl group that reacts with sulfhydryl groups at physiological pH. Crosslinking reactions with the water-insoluble NHS-esters are typically performed with a solvent carryover of 0. they can be generated by reduction of disulfide bonds. Maleimide reaction scheme. Alternatively. Sulfonated NHS-ester crosslinkers are supplied as sodium salts and are soluble in water to a concentration of at least 10 mM. Excess maleimides can be quenched at the end of a reaction by adding free thiols.5. The non-sulfonated forms of NHS-ester reagents are waterinsoluble and are first dissolved in water-miscible organic solvent. such as DMSO (Product # 20688) and DMF (Product # 20672. especially above pH 8. A protein in a complex mixture can be specifically labeled if it is the only one with a free sulfhydryl group on its surface. EDTA can be included in the coupling buffer to minimize oxidation of sulfhydryls. In the absence of free sulfhydryls. Imidazoles can react with iodoacetyl groups at pH 6.0. At neutral pH. Histidyl side chains and amino groups react in the unprotonated form with iodoacetyl groups above pH 5 and pH 7. If there are insufficient quantities of free sulfhydryls. SATA (Product # 26102). In some cases. Thermo Scientific Pierce NHS-ester Haloacetyl Crosslinkers. or to download product instructions. Maleimides do not react with tyrosines. Loss of activity may also occur when any of the lysine groups involved in binding a substrate (in the case of an enzyme) or an antigen (in the case of an antibody) are modified by the crosslinker. crosslinking proteins with NHS-esters may result in loss of biological activity that may be a result of conformational change of the protein when the NHS-ester crosslinker reacts with primary amines on the molecule’s surface. but at pH >8. or SPDP (Product # 21857). The water-soluble NHS-ester crosslinkers are used for cell-surface conjugation because they will not permeate the membrane.

Carbohydrate modification is particularly useful for antibodies in which the carbohydrate is located in the Fc region away from binding sites. allowing it to be coupled to the amino group in the reaction mixture. Hydrazide reaction scheme. so reversal of the conjugation is impossible without destroying the protein. but increasing the amount of EDC can compensate for the reduced efficiency. Aldehyde-containing molecule Hydrazide compound Hydrazone linkage Figure 11. Oxidation of a carbohydrate (cis-diol) to an aldehyde The hydrolysis of EDC is a competing reaction during coupling and is dependent on temperature. In the presence of excess crosslinker. Failure to react with an amine results in hydrolysis of the intermediate. pyridine2-thione is released and its concentration can be determined by measuring the absorbance at 343 nm (Figure 10). Tris.5 to 7. mild oxidation of sugar glycols using sodium meta-periodate will convert vicinal hydroxyls to aldehydes or ketones (Figure 11). The disulfide exchange can be performed at physiological pH. other carbohydrates in proteins (including antibodies) will be targeted. making it ineffective in two-step conjugation procedures without increasing the stability of the intermediate using N-hydroxysuccinimide. At 1 mM NaIO4 and a temperature of 0°C. Thermo Scientific Pierce Pyridyl Disulfide Crosslinkers. 19 .08 x 103 M-1 cm-1 ). a disulfide exchange occurs between the molecule's –SH group and the 2-pyridyldithiol group. Carboxylate containing molecule EDC o-Acylisourea reactive ester B. The reaction with hydrazides is faster than with amines. Carboxy termini of proteins can be targeted. An EDC byproduct is released as a soluble urea derivative after displacement by the nucleophile (Figure 12). (See Table 4 for available pyridyldithiol crosslinkers. Reaction efficiency can be monitored by determining the concentration of the released pyridine-2-thione by measuring the absorbance at 343 nm (molar extinction coefficient at 343 nm = 8. As a result. o-Acylisourea reactive ester Amide bond Urea Figure 12. Pyridyl disulfide reaction scheme. The O-acylisourea intermediate is unstable in aqueous solutions. EDC coupling reaction scheme. the yield of the reaction is similar at pH from 4. call 800-874-3723 or 815-968-0747. glycine and acetate buffers may not be used as conjugation buffers. regeneration of the carboxyls and the release of an N-unsubstituted urea. Product # 28390) is an effective carbodiimide reaction buffer.5. These reagents can be used as crosslinkers and to introduce sulfhydryl groups into proteins. Subsequent reaction with hydrazides results in formation of a hydrazone bond. although the reaction rate is slower.Pyridyl disulfides Pyridyl disulfides react with sulfhydryl groups over a broad pH range (optimal pH is 4-5) to form disulfide bonds and. A. making them useful for site-specific crosslinking. the oxidation is restricted to sialic acid residues. 22981) reacts with carboxylic acid group and activates the carboxyl group to form an active O-acylisourea intermediate. During the reaction. therefore. However. resulting in the formation of amide or hydrazone bonds. The bond that results is the same as a peptide bond. however.5 to 5 and requires only a few minutes for many applications. To order. as well as glutamic and aspartic acid side chains. At concentrations of 6-10 mM periodate. Table 4. EDC (Product # 22980. The crosslinking reaction is usually performed between pH 4.) Carboxyl-reactive chemistry Carbodiimides Carbodiimides couple carboxyls to primary amines or hydrazides. Sulfhydrylcontaining molecule Pyridyl disulfide compound Disulfide bond Pyridine 2-thione Figure 10. Carbodiimides are unlike other conjugation reactions in that no spacer exists between the molecules being coupled. Carbonyls do not readily exist in proteins. Phosphate buffers reduce the reaction efficiency of the EDC. 4-Morpholinoethanesulfonic acid (MES. The oxidation is performed in the dark at 0-4°C to prevent side reactions. polymerization is likely to occur because all proteins contain carboxyls and amines. conjugates prepared using these reagents are cleavable. pH and buffer composition. Outside the United States. This intermediate reacts with a primary amine to form an amide derivative. Reagent LC-SPDP Sulfo-LC-SPDP PDPH Reactivity Sulfhydryl/Amine Sulfhydryl/Amine Sulfhydryl/Carbohydrate Product # 21651 21650 22301 Carbonyl-/Glyco-reactive chemistry Hydrazides Carbonyls (aldehydes and ketones) react with hydrazides and amines at pH 5-7. contact your local branch office or distributor.

an NHS-ester has much greater stability in slightly acidic or near-neutral pH conditions. which are not generally required unless protein concentrations are very low. visit www. A bright camera flash works well with the nitro. Reactions can be performed in a variety of amine-free buffer conditions. Forms of aryl azide-reactive groups in photo-reactive crosslinking reagents. continued NHS (Product # 24500) or its water-soluble analog Sulfo-NHS (Product # 24510) is often included in EDC-coupling protocols to improve efficiency. If working with heterobifunctional photo-reactive crosslinkers. use buffers compatible with the chemically reactive portion of the reagent. Nonspecific chemistries Aryl azides Photo-reactive reagents are chemically inert reagents that become reactive when exposed to ultraviolet or visible light. Figure 14). Thiol-containing reducing agents (e. preventing photoactivation. The traditional photo-reactive groups in these reagents are aryl azides (Figure 13).and hydroxyl-substituted aryl azides.. 20 For more information. When an aryl azide is exposed to UV light. Phenyl Azide Hydroxyphenyl Azide Nitrophenyl Azide Tetrafluorophenyl Azide Figure 13.com/pierce . These reagents will reduce the azide functional group to an amine. an NHS-ester has a half-life of one to several hours. The chemical reaction is performed in subdued light with reaction vessels covered in foil. resulting in an NHS-activated site on a molecule. primary amines. pH and structure of the crosslinker..g. The photoactivation can be performed with a bright camera flash or ultraviolet hand-held lamp about one to two inches above the reaction vessels. Unsubstituted aryl azides may require ultraviolet light or numerous flashes. The NHS-ester formed and the carbodiimide’s O-acylisourea intermediate are amine-reactive. In water. or subsequent ring expansion to react with a nucleophile (e. or even days. depending on temperature. The latter reaction path dominates when primary amines are present in the sample.g. EDC-mediated coupling of molecules works well in many applications without the addition of NHS or Sulfo-NHS.Crosslinker Reactivities Carbodiimides. it forms a nitrene group that can initiate addition reactions with double bonds. it is often necessary to reduce the EDC amount when converting to an EDC/NHS system to prevent excessive crosslinking and possible precipitation. but O-acylisourea intermediate has a half-life measured in seconds in acidic or neutral pH conditions. DTT or 2-mercaptoethanol) must be avoided in the sample solution during all steps before and during photoactivation. or to download product instructions. EDC couples NHS to carboxyls. When a large excess of EDC is used without NHS.thermo. insertion into C-H and N-H sites. however.

21 . Thermo Scientific Pierce Aryl Azide Crosslinkers.Phenyl Azide UV Light Active Hydrogen (C-H) Insertion Nitrene Formation Ring Expansion Active Hydrogen (N-H) Insertion Dehydroazepine Intermediate Reactive Hydrogen Addition Reactions Nucleophile Figure 14. Table 5. call 800-874-3723 or 815-968-0747. Reactive Groups Reagent ABH ANB-NOS APDP APG ASBA BASED Mts-Atf-Biotin Mts-Atf-LC-Biotin NHS-ASA SANPAH SPB Sulfo-HSAB Sulfo-NHS-LC-ASA Sulfo-SAED Sulfo-SAND Sulfo-SANPAH Sulfo-SASD Sulfo-SBED Sulfo-SFAD Product # 21510 21451 27720 20108 21512 21564 33093 33083 27714 22600 23013 21563 27735 33030 21549 22589 27716 33033 27719 Photo-reactive Phenyl azide Nitrophenyl azide Hydroxyphenyl azide Phenyl azide Hydroxyphenyl azide Hydroxyphenyl azide Tetrafluorophenyl azide Tetrafluorophenyl azide Hydroxyphenyl azide Nitrophenyl azide Psoralen Phenyl azide Hydroxyphenyl azide Azido-methylcoumarin Nitrophenyl azide Nitrophenyl azide Hydroxyphenyl azide Phenyl azide Perfluoroaryl azide Other Groups(s) Hydrazide NHS Pyridyldisulfide Phenylglyoxal Amine Hydroxyphenyl azide Methanethiosulfonate/Biotin Methanethiosulfonate/Biotin NHS NHS NHS Sulfo-NHS Sulfo-NHS Sulfo-NHS Sulfo-NHS Sulfo-NHS Sulfo-NHS Sulfo-NHS/Biotin Sulfo-NHS To order. Outside the United States. Possible reaction pathways of aryl azide crosslinkers. contact your local branch office or distributor.

Diazirine Sulfo-NHS. Diazirine Protein 1 NH2 O N O O O N N Protein 2 UV 350 nm N2 Protein 2 Protein 1 H N O H Figure 15. enabling their use for extracellular protein crosslinking. Diazirine-based photocrosslinkers have better photostability than phenyl azide-based photocrosslinkers and are easily activated with long-wave UV light (330-370 nm).Crosslinker Reactivities Diazirines The succinimidyl-ester diazirine (SDA) reagents are a new class of crosslinkers that combine proven amine-reactive chemistry with an efficient diazirine-based photochemistry for photo-crosslinking to nearly any other functional group. Mechanism of NHS-ester diazirine crosslinking. Photoactivation of diazirine with long-wave UV light (330-370 nm) creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths. Photoactivation of diazirines with long-wave UV light (330-370 nm) creates reactive carbene intermediates.thermo. The NHS-ester diazirine derivatives (SDA. SDAD and Sulfo-SDAD also have a disulfide bond within the spacer that can be cleaved with reducing agents. ability to cleave the crosslinked proteins. Diazirine Sulfo-NHS. or to download product instructions. By contrast. Diazirine NHS. Protein 1 H N O N N Thermo Scientific Pierce Diazirine-based Photo-crosslinkers. The SDA crosslinkers include six compounds differing in spacer arm lengths. Diazirine NHS. Such intermediates can form covalent bonds through addition reactions with any amino acid side-chain or peptide backbone at distances corresponding to the spacer arm lengths. Sulfo-LC-SDA and Sulfo-SDAD contain negatively charged sulfonate groups that improve their water solubility and reduce cell membrane permeability. visit www. Diazirine Sulfo-NHS. These SDA reagents extend the efficiency and range of interactions that can be explored by this approach.com/pierce . Sulfo-SDA. N-hydroxysuccinimide (NHS) esters react efficiently with primary amine groups (–NH2) in pH 7-9 buffers to form stable amide bonds upon release of NHS. and presence or absence of a charged group for differential membrane permeability. LC-SDA and SDAD) lack a charged group and are membrane-permeable. Reagent SDA LC-SDC SDAD Sulfo-SDA Sulfo-LC-SDA Sulfo-SDAD Product # 26167 26168 26169 26173 26174 26175 Reactive Groups NHS. 22 For more information. This property makes them ideal for intracellular and intramembrane conjugations.

researchers in academia and industry look to link two or more molecules to form novel complexes with uniquely combined properties of the individual components. books are nonreturnable. This is a must-have resource when searching for methods that will facilitate the conjugation of molecules to new reagents or for novel applications. Sorry. S. 850 figures and illustrations and up-to-date references. (2008). It also discusses the latest advances in bioconjugation methods applied to nanotechnology and proteinprotein interactions. Because the Avidin-Biotin system can be used in so many ways. you’ll want to keep this handbook close at hand! Product # 1601676 / Free! To order.. Thermo Scientific Avidin-Biotin Technical Handbook Avidin-Biotin Products This reference guide brings together everything needed to biotinylate cellsurface proteins. contact your local branch office or distributor. 2nd ed. As the field of bioconjugation continues to grow. 1202 pages. 2nd Edition Bioconjugate Techniques is the best single source of comprehensive knowledge and techniques required to design and synthesize any bioconjugate. 1991.D. a recognized bioconjugation technology expert. Outside the United States. purify a biotinylated target. detect a biotinylated antibody and perform many other applications. Bioconjugate Techniques.S. Hardcover.... call 800-874-3723 or 815-968-0747. has written a second edition of the Bioconjugate Techniques book. 340 pages. Product # 20036 Hermanson. Inc. selection guides and a complete listing of available tools. It comprehensively covers every reagent system for bioconjugation in research. It provides a complete update and major expansion of the original work which has been established as the definitive reference in the field. It includes dozens of references along with protocols.Books and Free Technical Handbooks! Bioconjugate Techniques. 23 . Chemistry of Protein Conjugation and Cross-Linking An excellent crosslinking reagent reference for the expert and the beginner. and therapeutic development. Ph. Greg Hermanson. books are nonreturnable. This 1202-page book is an extremely valuable source of information with easy-to-understand strategies.. Sorry. Product # 15010 Wong. Elsevier Inc. troubleshooting tips. diagnostics. Published by CRC Press. G.T. Details the critical aspects of protein crosslinking and conjugate preparation and covers the full spectrum from protein-reactive groups to proteinnucleic acid crosslinking.

2.Thermo Scientific Pierce Crosslinkers at a Glance Product # 21510 22295 21451 27720 21512 21564 22331 22330 22323 22297 22298 22336 22337 21610 21615 21580 21590 21595 21581 21582 21600 22405 22423 20320 21525 20660 21666 21667 20700 21702 20593 22585 21655 21555 21658 20589 20665 22335 21578 77149 22980 22981 Abbreviation ABH AMAS ANB-NOS APDP* ASBA* BASED* BMB BMH BMOE BMPH BMPS BM(PEG)2 BM(PEG)3 BS G-d0 2 Chemical Name p-Azidobenzoyl hydrazide N -(α-Maleimidoacetoxy)-succinimide ester N -5-Azido-2-nitrobenzyloxy-succinimide N -(4-[p-Azidosalicylamido]butyl)3'-(2'-pyridyldithio) propionamide 4-(p-Azidosalicylamido)-butylamine Bis (β-[4-azidosalicylamido]ethyl) disulfide 1. ***** Carbonyl reactive.35 403.1 Å 8.0 Å 16.27 474.7 Å 12 Å 11.15 259.2. ** Trifunctional crosslinking reagent.4 Å 7.71 436.7 Å 11. visit www.11-Bis-Maleimidotriethyleneglycol Bis (sulfosuccinimidyl)glutarate-d 0 Bis (sulfosuccinimidyl)2.4 Å 11.2 446.9 Å 7.18 305.29 352.7 Å 7.36 206.28 312.8 Å 7.37 608.33 204. *** Trifunctional crosslinking reagent.09 245.6 Å 9. 24 For more information.3 Å 10.1 Å 5.43 360.com/pierce .3'-Dithiobis (sulfosuccinimidylpropionate) 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride 344.4 Å 21.70 6.17 273. Size 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 10 mg 10 mg 50 mg 10 mg 10 mg 100 mg 100 mg 50 mg 25 mg 25 mg 100 g 50 mg 1g 50 mg 1g 1g 50 mg 50 mg 1g 50 mg 1g 8 x 2 mg 50 mg 1g 50 mg 50 mg 10 mg 5g 25 g M.9 Å 14.4-Bis-Maleimidobutane Bis-Maleimidohexane Bis-Maleimidoethane N -(β-Maleimidopropionic acid)hydrazide•TFA N -(β-Maleimidopropyloxy)succinimide ester 1.2 Å 11 Å 19.7 Å 35.42 368.3 Å 21.N-Dicyclohexylcarbodiimide 1-5-Difluoro-2.45 532.35 Spacer Arm 11. Streptavidin or NeutrAvidin™ Protein.8-Bis-Maleimidodiethylene-glycol 1.4-glutarate-d 4 Bis (sulfosuccinimidyl)suberate Bis (sulfosuccinimidyl)suberate-d 0 Bis (sulfosuccinimidyl)2.26 404.20 482.4 Å 13.4.9 Å 16. or to download product instructions.4-dinitrobenzene Dimethyl adipimidate•2HCI Dimethyl pimelimidate•2HCI Dimethyl suberimidate•2HCl 1.W.5 708.51 191.52 248.4 Å – NH2 Amines X X X X X X X X X X X X X X X X X X X X X BS2G-d4 BS (Sulfo-DSS) 3 3 BS -d0 BS3-d4 BS[PEG]5 BS(PEG)9 BSOCOES C6-SANH***** C6-SFB****** DCC DFDNB DMA DMP DMS DPDPB DSG DSP DSS DST DTBP DTME DTSSP (Sulfo-DSP) EDC Disuccinimidyl tartarate Dimethyl 3.43 576.3'-dithiobispropionimidate•2HC Dithiobis-maleimidoethane 3.35 534.4 Å 11.55 249.21 308.4-Di-(3'-[2'pyridyldithio]propionamido) butane Disuccinimidyl glutarate Dithiobis(succimidylpropionate) (Lomant’s Reagent) Disuccinimidyl suberate Pkg.3 Å 12 Å 0Å X X X X * Crosslinker is iodinatable.0 Å 8. **** Reacts selectively with arginine at pH 7-8. binds to Avidin.5 Å 0Å 3Å 8.18 297.7 Å 17.71 326. 177.29 220.19 266.8 13 Å 14.43 572.24 309.38 572.7.23 276.7-suberate-d 4 Bis (NHS)PEG5 Bis (NHS)PEG9 Bis(2-[succinimidoxycarbonyloxy]ethyl)sulfone C6-Succinimidyl 4-hydrazinonicotinate acetone hydrazone C6-Succinimidyl 4-formylbenzoate N.9 Å 13.34 530.thermo.4 Å 11. ****** Hydrazine/Hydrazone reactive.16 252.7 Å 21.9 Å 4.

– SH Sulfhydryls X Carbohydrates X Nonselective (photo-reactive) X X – COOH Carboxyls –OH Hydroxyl Heterobifunctional X X X X Cleavable By X X X X X Thiols X Thiols X X X X X X X X X X Base X X X Thiols Thiols Periodate Thiols X X X Thiols Thiols To order. Outside the United States. call 800-874-3723 or 815-968-0747. contact your local branch office or distributor. 25 .

3 Å Atf-Biotin 35.3 Å 17.36 447.46 247. ****** Hydrazine/Hydrazone reactive.75 839.6 Å 8.com/pierce .1 Å 9.71 865.5 601.36 211. ***** Carbonyl reactive. or to download product instructions.8 Å 7.4 Å 95.68 283.32 214.33 * Crosslinker is iodinatable.5.23 281.thermo. Streptavidin or NeutrAvidin™ Protein.2 11.14 334. visit www.10 225.4 Å 11. binds to Avidin.35 295.20 388.48 425. 26 For more information.5 16. ** Trifunctional crosslinking reagent.6 689.5 39.2 Å 8.02 402.55 356.5 5. Size 1g 1g 50 mg 50 mg 50 mg 100 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 5 mg M. *** Trifunctional crosslinking reagent.9 Å Mts-Atf 11.7 Å 19.7 Å 7.25 309.38 338. **** Reacts selectively with arginine at pH 7-8.0 Å 12.2 Å 15.35 307.18 290.4 Å 7.6-tetrafluorobenzoyl)-N6(6-biotinamidocaproyl)-L-lysinyl]ethylmethanethiosulfate 2-{N2-[N6-(4-Azido-2.20 311.21 229.5.2 Å 3.3 ÅAtfBiotin 30.6-tetrafluorobenzoyl)-N6(6-biotinamidocaproyl)-L-lysinyl]}ethylmethanethiosulfate N-Hydroxysuccinimidyl-4-azidosalicylic acid 3-(2-Pyridyldithio)propionylhydrazide N-(p-Maleimidophenyl)isocyanate Succinimidyl 4-hydrazinonicotinate acetone hydrazone N-Succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate Succinimdyl 3-(bromoacetamido)propionate NHS-Diazirine NHS-SS-Diazirine Succinimidyl 4-formylbenzoate Succinimidyl 4-hydrazidoterephthalate hydrochloride N-succinimidyl iodoacetate N-Succinimidyl(4-iodoacetyl)aminobenzoate Succinimidyl 4-(N-maleimido-methyl)cyclohexane-1carboxylate NHS-PEG2-Maliemide NHS-PEG4-Maliemide NHS-PEG6-Maleimide NHS-PEG8-Maliemide NHS-PEG12-Maliemide NHS-PEG24-Maleimide Succinimidyl 4-(p-maleimido-phenyl)butyrate Pkg.6 Å 24.8 Å 9.9 13.95 Spacer Arm 16.1 Å Mts-Biotin 29.39 513.3.2 Å 53. 456.92 1394.W.7 Å 18.Thermo Scientific Pierce Crosslinkers at a Glance Product # 21565 22306 22106 22308 22309 22211 22111 26168 22362 21651 22311 22305 33093 Abbreviation EGS EMCA EMCH EMCS GMBS KMUA KMUH LC-SDA LC-SMCC LC-SPDP MBS MPBH Mts-Atf-Biotin** Chemical Name Ethylene glycol bis(succinimidylsuccinate) N-ε-Maleimidocaproic acid N-(ε-Maleimidocaproic acid)hydrazide N-(ε-Maleimidocaproyloxy)succinimide ester N-(γ-Maleimidobutyryloxy)succinimide ester N-κ-Maleimidoundecanoic acid N-(κ-Maleimidoundecanoic acid)hydrazide NHS-LC-Diazirine Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate) Succinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate m-Maleimidobenzoyl-N-hydroxysuccinimide ester 4-(4-N-Maleimidophenyl)-butyric acid hydrazide•HCI 2-[N2-(4-Azido-2.24 308.7 Å Mts-Atf 21.7 Å 6.11 27714 22301 28100 22400 22600 22339 26167 26169 22419 22411 22349 22329 22360 22102 22103 22104 22107 22105 22108 22112 22113 22114 22416 NHS-ASA* PDPH PMPI SANH***** SANPAH SBAP SDA SDAD SFB****** SHTH***** SIA SIAB SMCC SM[PEG]2 SM[PEG]4 SM(PEG)6 SM[PEG]8 SM[PEG]12 SM(PEG)24 SMPB 50 mg 50 mg 50 mg 25 mg 50 mg 50 mg 50 mg 50 mg 100 mg 25 mg 50 mg 50 mg 50 mg 100 mg 1g 100 mg 1g 100 mg 100 mg 100 mg 1g 102 mg 50 mg 276.32 425.29 280.21 225.3 Å 17.27 390.5 Å 10.2 Å 8.3.2 Å 6.0 Å 9.52 314.6 Å X X X X X X X X X X X X X X X X X X X X X X X X X – NH2 Amines X X 33083 Mts-Atf-LC-Biotin** 5 mg 953.6 Å 32.9 Å 1.8 Å Mts-Biotin 29.3 Å 15.

contact your local branch office or distributor. Outside the United States. 27 .– SH Sulfhydryls X X X X X X X X X X X Carbohydrates Nonselective (photo-reactive) – COOH Carboxyls –OH Hydroxyl Heterobifunctional X Cleavable By Hydroxylamine X X X X X X X X X X X X Thiols X X X Thiols X X Thiols X X X X X X X X X X X X X X X X Thiols Thiols X X X X X X X X X X X X X X X X X X X X To order. call 800-874-3723 or 815-968-0747.

0 Å 16.3 Å 18.15 386.3 Å 4.6 Å 10.4 Å 7.3'-dithiopropionate Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate β-(Tris[hydroxymethyl]phosphine)propionic acid (betaine) Tris-(2-Maleimidoethyl)amine (Trifunctional) Tris-(succimimidyl aminotricetate) (Trifunctional) 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 327.03 Å 10.36 * Crosslinker is iodinatable.36 482.2 Å X X X X X X X X X X X X X X X X X X X X X Pkg. ****** Hydrazine/Hydrazone reactive. **** Reacts selectively with arginine at pH 7-8. visit www.37 458.6 Å 3.33 382.W.1 Å 3.19 436.5-9.3'-dithiopropionate Sulfosuccinimidyl 6-(4'-azido-2'nitrophenylamino)hexanoate Sulfo-NHS-(2-6-[Biotinamido]-2-(p-azidobezamido) 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 5 mg 50 mg 50 mg 10 mg 660.com/pierce .48 504.46 385.3 Å 9.28 362.6 570.57 416. ** Trifunctional crosslinking reagent. binds to Avidin.38 197.32 312. 28 For more information.30 491.40 603. ***** Carbonyl reactive.5 20.51 492.3 Å 11.1 Å Biotin 19.3'-dithiopropionate Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido) ethyl 1.6 Å 18.7 Å 7.5 14.3 Å 12.36 388. 379.9 13. or to download product instructions.5 Å 6. Streptavidin or NeutrAvidin™ Protein.98 16.51 597.37 Spacer Arm 14.8 Å – NH2 Amines X X X X 26173 26175 27719 22327 22322 22317 22607 33043 33063 Sulfo-SDA Sulfo-SDAD Sulfo-SFAD Sulfo-SIAB Sulfo-SMCC Sulfo-SMPB THPP TMEA*** TSAT*** Sulfo-NHS-Diazirine Sulfo-NHS-SS-Diazirine Sulfosuccinimidyl(perfluoroazidobenzamido) ethyl 1.41 621.45 410. *** Trifunctional crosslinking reagent.25 490. Size 50 mg 50 mg 50 mg 50 mg M.0 Å 15.2 Å Sulfo-NHS ester 13.40 879.47 440.0 Å 8.67 527.0 Å 23.6 Å 8.1 Å 9.5Å 18.7 Å Phenyl azide 9.25 485.2 Å 20.thermo.Thermo Scientific Pierce Crosslinkers at a Glance Product # 22363 21558 23013 21857 21880 21566 22307 22324 21563 21111 26174 21568 21650 22312 27735 33030 21549 22589 33033 Abbreviation SMPH SMPT SPB SPDP Sulfo-EGS Sulfo-EGS Sulfo-EMCS Sulfo-GMBS Sulfo-HSAB Sulfo-KMUS Sulfo-LC-SDA Sulfo-LC-SMPT Sulfo-LC-SPDP Sulfo-MBS Sulfo-NHS-LC-ASA* Sulfo-SAED Sulfo-SAND Sulfo-SANPAH Sulfo-SBED** Chemical Name Succinimidyl-6-(β-maleimidopropionamido)hexanoate 4-Succinimidyloxycarbonyl-methyl-α-(2-pyridyldithio)toluene Succinimidyl-(4-psoralen-8-yloxy)butyrate N-Succinimidyl 3-(2-pyridyldithio)propionate See BS3 Ethylene glycol bis (sulfo-succinimidyl succinate) N-(ε-Maleimidocaproyloxy)sulfosuccinimide ester N-(γ-Maleimidobutryloxy)sulfosuccinimide ester N-Hydroxysulfosuccinimidyl-4-azidobenzoate N-(κ-Maleimidoundecanoyloxy)sulfosuccinimide ester Sulfo-NHS-LC-Diazirine Sulfosuccinimidyl 6-(α-methyl-α-[2-pyridyldithio]-toluamido) hexanoate Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]propionamido)hexanoate m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester Sulfosuccinimidyl(4-azido-salicylamido) hexanoate Sulfosuccimidyl 2-[7-azido-4-methylcoumarin3-acetamido]ethyl-1.

29 . Outside the United States.– SH Sulfhydryls X X Carbohydrates Nonselective (photo-reactive) – COOH Carboxyls –OH Hydroxyl Heterobifunctional X X Cleavable By Thiols X X X X Thiols Hydroxylamine X X X X X X X X X X X X X X X X X X X X X X X X X X X Thiols Thiols Thiols Thiols Thiols X X X X X X X X X X X X X Thiols Thiols To order. call 800-874-3723 or 815-968-0747. contact your local branch office or distributor.

Bioconjugate Techniques. G. p. Chem.27 Spacer Arm 16. J. J. (1996). San Diego: Academic Press. pp.M.T. 416. R. (1989). 474. 8604-8608. San Diego: Academic Press.52 Spacer Arm 21.. 27(10). 1325-35.18 Spacer Arm 4.C. 252.. U. 305. 416. Oxford: IRL Press. 177. 284.55 Spacer Arm 21. 21564 BASED M. or to download product instructions. 101.M. Bioconjugate Techniques. et al.thermo. Protein Function. (1996). 446.Appendix I – Structures Appendix I Structures Product # 21510 Product Name ABH M.W.T. 27720 APDP M.com/pierce .4 Å • May. • Sayre. Sci..W. et al. (1984). 21512 ASBA M.W. Proc.16 Spacer Arm 11. (1989).W. 21451 ANB-NOS M.3 Å • Hermanson.9 Å Structure Reference 22295 AMAS M. Med.7 Å • Krieg. L. et al. Natl. A Practical Approach.3 Å • Hermanson.W. visit www. 1718-1725. G.R.0 Å • Traut. (1986). Biochemistry 28. Acad. 249.20 Spacer Arm 7. USA 83. 214. pp. 30 For more information.W.

1 Å • Krieg. 18237-18243. Tokyo/New York.. Biol. 297. 266(27). (1991). L. Pharm. Biol. 31 .Appendix I Structures Product # 22331 Product Name BMB M.L. 266(27). 308. J. 3031-3039. J.W. T.. Chem. call 800-874-3723 or 815-968-0747.. 29(4). Proc. USA 83.W. L. et al. L. Acad.W. (1991). et al.W. 81-89. 22330 BMH M. Natl. 248. 22323 BMOE M. et al. 266. (1981). (1986)...34 Spacer Arm 17.21 Spacer Arm 5. (1991). 18237-18243. 1130-1135. 24(15).0 Å • Chen.W. J.1 Å • Chen. 220. 18237-18243.L. 8604-8608. T.9 Å • Kitagawa.A. Sci.9 Å Structure Reference • Chen. (1981). et al. Bull.19 Spacer Arm 8.29 Spacer Arm 16. Enzyme Immunoassay.29 Spacer Arm 14.7 Å 22337 BM(PEG)3 M.W. 266(27). 352. 22336 BM(PEG)2 M. Chem. Chem. 22298 BMPS M. Nucleic Acids Res. Biol.L.23 Spacer Arm 10.C. • Chrisey.W. L. 276. Igaku-Shoin pp. Outside the United States. contact your local branch office or distributor. et al. (1996). • Kitagowa. Chem.8 Å To order. et al. U.18 Spacer Arm 8.. 22297 BMPH M.

7 Å Bis (NHS)PEO5 O O O N O O M.45 Spacer Arm 11.7 Å O O O O O N O O 21615 BS 2G-d4 M.W.4 Å 21581 BS(PEG)5 M.71 Spacer Arm 35.4 Å 21595 BS3-d4 M.com/pierce . 530.5 Spacer Arm 21.W.7 Å O N O O O O [ [ O ]5 ]9 O O N O 21582 BS(PEG)9 M.43 Spacer Arm 11.45 Spacer Arm 11.W.W. 572.4 Å M.8 Å O N O O O M.W.W. Chem. 532.43 Spacer Arm 11. 534..4 Å BS G-d4 • Knoller. 708. 572. et al. (1991). S. 576.thermo.50 Spacer Arm 21.W. 576.W.38 Spacer Arm 7.Appendix I – Structures (continued) Appendix I Structures Product # 21610 Product Name BS G-d0 2 Structure O S O N O – O Na+ Reference Na+O – O S O M. Biol. or to download product instructions. 708.W. visit www. 21590 BS3-d0 M.35 Spacer Arm 7.7 Å O S O O–Na+ O O N O O D D 2 Na+O O O N O – O S O O D D 21580 BS3 (Sulfo-DSS) M.71 Spacer Arm 35. 266. 532. 2795-2804. J.8 Å BS(PEG)9 32 For more information.W.

W. 259. J. 10766-10769. Biochem.W.5 Å 20320 DCC M. To order.A. 436. Biochemistry 6(8).6 Å • Hartman. S. 245. et al.W.36 Spacer Arm 13. call 800-874-3723 or 815-968-0747. 913-920.W. J. 360. D. Biol. D. F. (1967). Biochemistry 16(13). (1977). Outside the United States.F. 124.09 Spacer Arm 3.20 Spacer Arm 11. • Zarling.35 Spacer Arm 13. 155. 273. 206.0 Å • Wang. and Lake.A. Z. 257(18).. 33 . et al. D. 219-224.4 Å 22423 C6-SFB M. 20700 DMS M. F.17 Spacer Arm 9. (1986). J.0 Å • Kornblatt. 403.W. J. (1980).. (1982). C. and Wold. 141-147. 22405 C6-SANH M. Immunol.2 Å • Schneider.W.15 Spacer Arm 8. 21666 21667 DMP M. 58. Can J. Chem. Eur. 2937-2942.C.Appendix I Structures Product # 21600 Product Name BSOCOES M. et al.W.33 Spacer Arm 0 Å 21525 DFDNB M.W.0 Å Structure Reference • Bouizar.. 2439-2448. 204.43 Spacer Arm 14. Biochem. and Moore. 20660 DMA M. contact your local branch office or distributor. (1980).

608. 168-175. M. 312. J.W.thermo. 326.W. 3448-3455. • Park.. Immunol. 1719-1726. J.W. and Atkinson. J.4 Å • Farries. 205-210.W. 261. and Moroi. S.A.W. et al. (1989).4 Å • Cox. Biochemistry 28. (1991). 22335 DTME M. et al. R.Appendix I – Structures (continued) Appendix I Structures Product # 21702 Product Name DPDPB M. (1989).28 Spacer Arm 11. 20589 DST M. (DST example) 20665 DTBP M. 21578 DTSSP (Sulfo-DSP) M.9 Å • Shivdasani. 142..0 Å • Joshi. S. 145. • Han. 344.7 Å • Waugh. Biol. R.Y.. and Thomas. Anal. and Han.51 Spacer Arm 12. (1988). et al. Biochem. (1994). (1995). Immunol. Acta 761. Chem. J. L.. Biol. S. 482. (1983). et al. D. 21655 21555 21658 DSS M. Anal. Biochim.9 Å Structure Reference • Chen. and Burrows.42 Spacer Arm 12. 34 For more information. J. G. 309. 265. T.com/pierce .P.C.W.26 Spacer Arm 7.L. 368. (1990).35 Spacer Arm 11.W.W. 842-847. Immunol. Chem. L. 1252-1260. L.W. 5-10.0 Å • Jung. Biophys.M.L. 141. 266(27). (1990). (1986). 220. 14518-14525. Biochem. 404. J. (EGS example) 22585 DSP M. 20593 DSG M. et al.M. Biol.S. G.37 Spacer Arm 13.C..71 Spacer Arm 19. 18237-18243. or to download product instructions. 227.W. J. J. visit www. Chem.3 Å • Chen.24 Spacer Arm 6. 152-162.

J. 134. et al. et al.24 Spacer Arm 11. (1975).. Immunol.) 22309 GMBS M.0 Å • Trail.8 Å • Trail.. 225. 1004-1010. 295. Biopolymers 22(1). 77-83. Sci.W. • Browning. 265. 185(1). Anal.W. Proc. J. 22106 EMCH M. To order. 133-143. (1986). 261-263. Methods 120. (1996).G. Methods 112. 77-83.. et al. Science 261. 456.W. J.A. (1990). Chem. (1983). Outside the United States.. 191. (1996). 281.23 Spacer Arm 7. • Peeters. (1988).7 Å • Rich. Immunol. 481-486. L. M. (1993). 1859-1867.B..W. P. San Diego: Academic Press. (1993).4 Å • Fujiwara. Biochem.H. • Hermanson. Acad. 18. 212-215. 280. et al. and Ribolini. (1990).. and Rozengurt. 24(15). Methods 112.M. 22111 KMUH M.W. A.. et al.. call 800-874-3723 or 815-968-0747. 211. • Grabarek. 131-135. (1981). et al.W. 4094-4098.3 Å • Fujiwara.. contact your local branch office or distributor.. G. Immunol.1 Å • Millar. J. and Gergely. Biol. 35 . Science 261. Z. K. K. et al.70 Spacer Arm 0 Å Structure Reference • Taniuchi. • Moroder. J. (1988). FEBS Lett. Immunol. 212-215. Natl.W. J. • Chrisey.21 Spacer Arm 9. Nucleic Acids Res. P. (1989). 22308 EMCS M.W. (EGS example) 22306 EMCA M.A. D. 308. (1989). Med.35 Spacer Arm 15. et al. L.A. 3031-3039. E. Bioconjugate Techniques. USA 83. 143. J.Appendix I Structures Product # 77149 22980 22981 Product Name EDC M. 22211 KMUA M.36 Spacer Arm 16.T.29 Spacer Arm 9.4 Å • Griffith. J. 12052-12058. 243-245. et al.38 Spacer Arm 19. (EMCS use can be modeled after GMBS. J. Chem. et al. 21565 EGS M. J. D.

9 Å • Chamow. et al. Methods 138. Nucleic Acids Res. J. J. Immunol. T. 425. 101. 338.. J. 314. 723-737. and Aikawa.com/pierce .75 Spacer Arm 17.W. (MBS example) • Chrisey. 21651 LC-SPDP M.M. et al.. 129-142. 3031-3039. or to download product instructions. 447.. • Yoshitake. Eur.W.E. 233-236. L. 7. Biochem.. Methods 121. 112. Chem. et al..7 Å • Cumber.W. (Tokyo) 79.W. • Carlsson. (1979). 24(15). (1989). (1976). 207-225. 22311 MBS M. et al.W. 88-95.. (1992).. J. 15916-15922. (1996). et al. 87-94. et al. I. J. C. J.95 36 For more information. (1991). D. 173.A.3 Å • Kitagawa.25 Spacer Arm 7. (1978). 267(22).36 Spacer Arm 12.W. Methods Enzymol.52 Spacer Arm 15. (1996). A.2 Å • Uto.5 Å O N O O O H N N N Structure Reference NHS-LC-Diazirine Spacer Arm 12. Chem.. Bioconjug. T.thermo.J. visit www. Immunol. 33093 Mts-Atf-Biotin Mww Mts-Atf-Biotin C32H45F4N907S3 M. 309. Biochem.Appendix I – Structures (continued) Appendix I Structures Product # 26168 Product Name LC-SDA M. • Bieniarz. et al. et al. Biochem. 839. 22305 MPBH M. Biol. • Myers. 395-399.48 Spacer Arm 16. (1985). S. J. S.5 Å LC-SDA O 22362 LC-SMCC M.

S. et al. • Zara. J. Chem. G.11 Spacer Arms Mts-Atf 21. Outside the United States. et al.2 Å • Greenfield.7 Å • Annunziato. J. 1243-1247.E. (1993). et al. (1990).S.2 Å • Wood. et al. 953. R. (1993). Chem. Biochem. 6600-6607. 260. et al. M. 953. Bioconjug.. 4. 265(19). 290.0 Å • van der Horst. (1991)..35 Spacer Arm 18. 37 ..L. 214.W.11 27714 NHS-ASA M. (1985).27 Spacer Arm 6.3 Å Atf-Biotin 35.J.W. 229.W. J.W. Anal.M. (1990). 50. 390. (HSAB example) To order.8 Å Mts-Biotin 29. 10801-10804. M. Biol.W.21 Spacer Arm 8.Appendix I Structures Product # 33083 Product Name Mts-Atf-LC-Biotin M. call 800-874-3723 or 815-968-0747. 28100 PMPI M. C.W.2 Å Structure Reference Mts-Atf-LC-Biotin C38H56F4N1008S3 M. (NHS-ASA example) 22301 PDPH M.18 Spacer Arm 8. 212-218. Chem.. Biol. Cancer Res.J. 373-377.32 Spacer Arm 9. • Friden. 156-162. 194..7 Å 22600 SANPAH M. Science 259. 276. 22400 SANH M. contact your local branch office or distributor. P.W. and O’Dorisio.T.

63-71.3 Å M.14 Spacer Arm 10.32 Spacer Arm 8.8 Å 22411 SHTH M.W.S.E.W.W. et al. (1996). J.com/pierce . Bioconjugate Techniques. or to download product instructions. M. 334.W. visit www.W. Chem.9 Å O N O O O Structure N N Reference NHS-Diazirine Spacer Arm 3. 307. 321-336.10 Spacer Arm 6.. J. (1985).T.. P.9 Å SDA 26167 SDAD M.68 Spacer Arm 7.10 Spacer Arm 6. 307. Biochem 140. et al. 568.5 Å 22329 SIAB M. Eur. 542. 112. 225. 283. 207-225.W.46 Spacer Arm 13.02 Spacer Arm 1.2 Å 22419 SFB M. (1978). 553.32 Spacer Arm 11.W. 458-463. E. Methods Enzymol.02 Spacer Arm 1.6 Å SMCC 38 For more information.Appendix I – Structures (continued) Appendix I Structures Product # 22339 Product Name SDA M. Immunol. San Diego: Academic Press.W. Methods 24. (1991). 311. 22360 SMCC M.J.20 Spacer Arm 3. et al.thermo.6 Å • Cumber.5 Å S S H N O N N SDAD 26169 SBAP M. A. J.2 Å SBAP • Inman.W. Bioconjug.W.5 Å O N O O O NHS-SS-Diazirine Spacer Arm 13. pp. 283. G. 402. (1984)..K.. 2. 247. et al. 388. • Rector.W.20 Spacer Arm 5.5 Å SIA • Thorpe. M. • Hermanson.9 Å 22349 SIA M. 334.

Biochem 140.6 Å Spacer Arm 17. 1394.2 Å SM(PEG)24 [ ] 22416 SMPB M. E.4 Å SM(PEG)12 [ ] 22114 SM(PEG)24 M.689.33 Spacer Arm 11. 601.W. 356.50 Spacer Arm 24.E. • Rector. 356. J.5 Spacer Arm 24.6 Å SM(PEG)4 [ ] 22105 SM(PEG)6 M.W.2 Å M.W.60 Spacer Arm 32.W. 513..W.2 Å M.W. 1394. call 800-874-3723 or 815-968-0747. Outside the United States. J.Appendix I Structures Product # 22102 22103 Product Name SM(PEG)2 SM(PEG)2 Structure Reference M.5 Å M.36 Spacer Arm 14. Immunol. Eur.W. et al. M.33 Spacer Arm 11.W.W. 865.2 Å M.6 Spacer Arm 32.W.6 Å SMPB • Thorpe.6 Å 22104 22107 SM(PEG)4 M. et al. Methods 24.6 Å M. 865.71 Spacer Arm 39.5 Å SM(PEG)6 [ ] 22108 SM(PEG)8 M.71 Spacer Arm 39. 601. 39 .W.39 M. P.36 Spacer Arm 14. (1978).2 Å SM(PEG)8 [ ] 22112 22113 SM(PEG)12 M.92 Spacer Arm 53.S.W. 321-336.39 Spacer Arm 17.W. contact your local branch office or distributor. (1984). 63-71.92 Spacer Arm 53.55 Spacer Arm 95. 425. 379.W.4 Å M. 513.3 Å SMPH To order. 425. 689.55 Spacer Arm 95.6 Å 22363 SMPH M.W.. 379.W.

J. J. 382. D. (1988). 133-143. Bioconjug.. Acad. J.. 1859-1867.W. Biol.W.. Immunol. 24-31. and Schnos. J.6 Å • Laskin.W.D. Methods 112. • Hermanson. • Wang. 23013 SPB M. 660. et al.. Methods 112. and Ribolini. 40 For more information. et al.25 Spacer Arm 9. Chem. J. 21566 Sulfo-EGS M. Bioconj. A.W. Biochem. et al. 77-83. 362. 1.com/pierce .. Mol. 5. • Elsner. 77-83.. K. (1996). (1986). J. (1988). 143. 243-245. (1990). J.. 723-737. et al. J.47 Spacer Arm 16. Chem. 21563 Sulfo-HSAB M.thermo.T.3 Å • Fujiwara. Proc. J. Chem.33 Spacer Arm 9.W. G. J. Immunol. (1989).W.32 Spacer Arm 8. et al. V.B.37 Spacer Arm 6.0 Å Structure Reference • Ghetie.1 Å • Browning. (1987). et al. Natl.46 Spacer Arm 20. Bioconj. 377-384. M. Bioconjugate Techniques.W. 8.4 Å • Fujiwara. visit www. • Inman. R. K. (1994). Sci. 878-884.0 Å 21111 Sulfo-KMUS M. 410. 8211-8215.28 Spacer Arm 7. (1978). 388.45 Spacer Arm 16. Methods 112.) 22324 Sulfo-GMBS M. and Mouritsen. (1989). San Diego: Academic Press. et al. 173. (EMCS use can be modeled after GMBS. • Peeters. USA 83. Immunol. Immunol. 193. 77-83.3 Å • Fujiwara.M. 21857 SPDP M. et al. J. 385. K. 312.W. S.8 Å • Carlsson. (EGS example) 22307 Sulfo-EMCS M.I. or to download product instructions. Immunol. 480. (1988).. H. 463-467. Methods 120. (1997).Appendix I – Structures (continued) Appendix I Structures Product # 21558 Product Name SMPT M.

J.. 440. B. 621. call 800-874-3723 or 815-968-0747. (1996).41 Spacer Arm 18. Immunol.25 Spacer Arm 3. (1997).T. J. et al. Methods 121. 527. J. G.7 Å • Carlsson. Bioconjugate Techniques. et al.30 Spacer Arm 7.J.W.W. 327.W. (1989). 59.E. 491.T.. (NHS-ASA example) 33030 Sulfo-SAED M. 26173 Sulfo-SDA M. 723-737.51 Spacer Arm 13.W. 358a. 22312 Sulfo-MBS M.5 Å Sulfo-SDAD To order. J.W.57 Spacer Arm 15. Biochem. 21650 Sulfo-LC-SPDP M. 603. 41 . 129-142.W. (1991). Biol. San Diego: Academic Press. et al.B. Chem.9 Å Sulfo-SDA 26175 Sulfo-SDAD M. 173.W. 416. 935-940.60 Spacer Arm 23. G. S. et al.W. (MBS example) 27735 Sulfo-NHS-LC-ASA M. 265(19).5 Å Structure Reference 21568 Sulfo-LC-SMPT M. • Rajur.40 Spacer Arm 12. D.3 Å • Myers.0 Å • van der Horst. Biophys. contact your local branch office or distributor. J. Chem. Bioconjug.. 8. (1990). et al. Outside the United States. 232-235.5 Å Sulfo-NHS-SS-Diazirine Spacer Arm 13.67 Spacer Arm 20.9 Å Sulfo-NHS-Diazirine Spacer Arm 3.0 Å • Hermanson.. (1978).. 10801-10804.6 Å • Thevenin.Appendix I Structures Product # 26174 Product Name Sulfo-LC-SDA M. 490. pp.

et al.6 Å O Na+ O– S O O O N O O S S N H F F O F F Structure Reference • Pandurangi. et al. 289. 1092-1100. Biol. Chem. San Diego: Academic Press. Chem. (2000). Biol. Biol.Appendix I – Structures (continued) Appendix I Structures Product # 27719 Product Name Sulfo-SFAD M.thermo. 1243-1247. • Trotman. 122. (1977).R.98 Spacer Arms Sulfo-NHS ester 13.S. M. G. 597. 570.51 Spacer Arm 18. R. • Kleene. (2000). R850-857. et al.J.L. (HSAB example) 33033 Sulfo-SBED M. D.. (1985). 275(6). R. K.. (2000).A. M. Biochemistry 16. Y. or to download product instructions.K. J. 5650-5654. et al.. (1996). J. R.7 Å Phenyl azide 9. 22327 Sulfo-SIAB M. 275(12). J. 208-221.V. Biol. and O’Dorisio.W. Chem. (1996). Cell. Biochemistry 39.48 Spacer Arm 14. pp. (ANB-NOS example) 22589 Sulfo-SANPAH M. (2001).6 Å SFAD • Lewis. Chem. 492. J.. J. Science 279. et al.2 Å • Wood. Biol. • Ilver. C.1 Å • Hermanson.W.. S. Bioconjugate Techniques. Am. K. et al. Bioconjugate Techniques. et al.T. 9893-9900. J.40 Spacer Arm 18.W.6 Å • Hermanson. G.D.19 Spacer Arm 10. 879. N N+ N– 21549 Sulfo-SAND M.W. R. (2000). (2001). Photobiol.1 Å Biotin 19.5 Å M. Biology 10(23). 1626-6127. 291. Nature Cell Biology 3.48 Spacer Arm 14. et al.C.. 3767-3771. (1997). Photochem.. et al. Curr. pp..T. 260.W.S. 65(2). Chem.W. • Geselowitz. 42 For more information. 9055-9061. • Alley. (2000). 22(6). Mol. 373-377. San Diego: Academic Press...C. 1615-1625. Bioconjug. Chem. L. 597. S1-S2. (1998). 239-242. (1995). (2002). 502-506. • Neely. D. 375. • Minami. and Neumann. 6(4).. • Horney. • Sharma. 2880-2889.com/pierce . et al. Soc. 504. • Daum. visit www. et al.E. 276(4).

580-581.38 Spacer Arm 11. Anal. H. 1863-1864. Mol. 386. Cell. J.W. J.W... et al. et al. • Teale.2 Å To order. 197. Chem. Chem.W.3 Å 33063 TSAT M. 43 . Outside the United States. Chem... 40. 2(1). (1994).6 Å • Iwai.J.03 Å • Berning. Antibody.H. 458. 219-225.15 Spacer Arm 3. 482. (1996). M. • Katti.3 Å Structure Reference • Samoszuk. Am. Soc. (1988). 277-282. 2.E.W. 2181-2182. 121(8).M..W. Immunol. call 800-874-3723 or 815-968-0747.K. W. Soc.36 Spacer Arm 4. 33043 TMEA M.36 Spacer Arm 10. J.. et al. • Petach. 22317 Sulfo-SMPB M. • Diagle. (1986). (1970). Current Science 70(3). D. 283-292. K. Chem. Commun. Immunoconjugates Radiopharm. and Kearney. 1658-1664..F. et al.. Biochem. contact your local branch office or distributor. J.37 Spacer Arm 8. Soc. (1994). et al. 22607 THPP M. 436. (1989). J. Commun. D. • Henderson. Chem.Appendix I Structures Product # 22322 Product Name Sulfo-SMCC M. (1999). 37-46. 171.V. J. Textile Res. et al. J. K.

The interactive selection guide will guide you through the process of choosing the appropriate crosslinker for your application.com/pierce. 44 For more information.com/pierce .thermo. or to download product instructions. choose “selection guides” from the Technical Resources drop-down menu and then choose the crosslinker selection guide.thermo.Appendix II – Online Interactive Crosslinker Selection Guide We have developed an interactive crosslinker selection guide to aid in deciding which crosslinker is the best for your application. visit www. Go to www.

At pH > 11. Pyridyl disulfide: Aromatic moiety with a disulfide attached to one of the carbons adjacent to the nitrogen in a pyridine ring. Yields are generally low with many different crosslinked products formed. Aryl azide: Compound containing a photoreactive functional group (e. contact your local branch office or distributor. Pyridine 2-thione is released when this reagent reacts with a sulfhydryl (–SH)-containing compound. Sulfhydryl: –SH groups present on cysteine residues in proteins. Spacer arm: The part of a crosslinker that is incorporated between two crosslinked molecules and serves as a bridge between the molecules. The ε-amine in lysine and N-terminal amines are the targets in proteins. chlorine or bromine attached to an acyl group on the molecule.. and crosslinking can be reversed. formed in proteins through –SH groups from two cysteine molecules. They are specific for primary (–NH2) amines between pH 7-9. therefore. NHS: Abbreviation for N-hydroxysuccinimide. These reagents do not result in the formation of a cross-bridge and have been termed zerolength crosslinkers. resulting in the loss of the halogen and the appearance of a characteristic color. Crosslinker: A reagent that will react with functional groups on two or more molecules to form a covalent linkage between the molecules. Hapten: A molecule recognized by antibodies but unable to elicit an immune response unless attached to a carrier protein. Homobifunctional crosslinker: Reagent with two identical reactive groups used to link two molecules or moieties. but are generally the most effective at neutral pH. Conjugation reagent: A crosslinker or other reagent for covalently linking two molecules. The amidine bond is protonated at physiological pH. call 800-874-3723 or 815-968-0747. Hydrophilic: Substances that readily dissolve in water. Polymer: A molecule composed of many repeating monomers. Moiety: An indefinite part of a sample or molecule. these are good nucleophiles that may be targeted for crosslinking. Carbodiimide: Reagent that catalyzes the formation of an amide linkage between a carboxyl (–COOH) group and a primary amine (–NH2) or a hydrazide (–NHNH2).g. iodoacetyl) that targets nucleophiles. Diazirine crosslinker: The succinimidyl-ester diazirine (SDA) crosslinkers combine amine-reactive chemistry with an efficient diazirine-based photochemistry for photo-crosslinking to nearly any other functional group. Substrate: A substance upon which an enzyme acts. it carries a positive charge. Outside the United States. These esters are subject to hydrolysis. These alkylating reagents degrade when exposed to direct light or reducing agents. Oligomer: A molecule composed of several monomers. Integral membrane protein: Protein that extends through the cell membrane and is stabilized by hydrophobic interactions within the lipid bilayer of the membrane. N-Hydroxysuccinimidyl (NHS) ester: Acylating reagents commonly used for crosslinking or modifying proteins. The photoactivation of diazirine with long wave UV light (330-370 nm) creates carbene intermediates. Imidoester: Amine-reactive functional group of an imidate crosslinker. but not always.. α-Haloacyl: Functional group (e. which react so quickly and broadly that specific groups are not easily and efficiently targeted.Appendix III – Glossary of Crosslinking Terms Acylation: Reaction that introduces an acyl group (-COR) into a compound.g. Nonspecific crosslinking: Another term for nonselective crosslinking. sulfhydryls or –SH groups. These bonds often link polypeptide chains together within the protein and contribute to a protein’s tertiary structure. the amidine bond is unstable. with half-lives typically around 10-15 minutes at room temperature and pH 7-9. Immunogen: A substance capable of eliciting an immune response. Disulfide bonds: Oxidized form of sulfhydryls (. a protein that binds to a receptor. α-Haloacyl compounds have a halogen atom such as iodine. such as nitrenes or aryl azides. Ligand: A molecule that binds specifically to another molecule. Ultraviolet: Electromagnetic radiation of wavelengths between 10-390 nm. Imidates react with amines in alkaline pH conditions (pH range 7. Hydrophobic: Substances with limited solubility in water. phenyl azide) that reacts nonspecifically with target molecules. Thiols: Also known as mercaptens. Heterobifunctional crosslinker: Reagent with two different reactive groups used to link two molecules or moieties. Imidate crosslinker: Primary amine-reactive functional group that forms an amidine bond. small (< 5 kDa) molecules. These intermediates can form covalent bonds via addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths.S–S -). with half-lives approximating one to two hours at room temperature at neutral pH. Nitrene: Triple-bonded nitrogen-to-nitrogen reactive group formed after exposure of an azido group to UV light.5-10) and hydrolyze quickly. To order. thiolanes. Haptens are usually. Monomer: Consisting of a single unit. Its reactivity is nonspecific and short-lived. For example. 45 . especially thiols. Photoreactive: A functional group that becomes reactive upon excitation with light at a particular range of wavelengths. Nonselective crosslinking: Crosslinking using a reactive group.

com www.thermo.thermo.Contact Information Belgium and Europe. Unless indicated otherwise on the inside back cover.com www.com/pierce © 2009 Thermo Fisher Scientific Inc.euromarketing@thermofisher.com/perbio United States Tel: 815-968-0747 or 800-874-3723 Customer Assistance E-mail: Pierce. all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. These products are supplied for laboratory or manufacturing applications only. All rights reserved.CS@thermofisher. the Middle East and Africa Distributors Tel: +32 53 85 71 84 France Tel: 0 800 50 82 15 The Netherlands Tel: 076 50 31 880 Germany Tel: 0228 9125650 United Kingdom Tel: 0800 252 185 Switzerland Tel: 0800 56 31 40 Email: perbio. 1601673 04/09 .