EXTRACTION OF PROTEIN

The first step in the extraction of protein is liberation of protein from the cells that contain it and to get it in to solution. The method of choice for this purpose depends upon mechanical chracterstics of the source tissue and the location of protein in the cell. If protein of interest is located in to cytosol, its liberation requires only breaking open of the cells or lysis of the cells. A common method of lysis is to place the cells in hypotonic solution. Under the influence of osmotic forces, water enters the cell and lyses it. This method of lysis is known as osmolysis. Although this method works well with animal cells, but with bacterial and plant cells, which have cell wall, this method is ineffective. The use of an enzyme such as lysozyme which chemically degrades the bacterial cell wall is useful for these cells. Detergents or organic solvents such as acetone or toluene are also useful in lysing the cells but care must be exercised in their use as they may denature the protein of interest. Many cells require some sort of mechanical disruption process to break them open. This may include grinding with sand or alumina, the use of high speed blender, a homogenizer.or sonication ( breaking open cells through the use of ultrasonic vibrations) Once the cells have been lysed , the crude lysate may be filtered or centrifuged to remove the particulate cell debris, thereby leaving the protein of interest in supernatant solution. If the required protein is a component of subcellular assemblies such as membranes or mitochondria, that subcellular fraction is first separated and isolated by differential centrifugation. Differential centrifugation is a process by which the cell lysate is centrifuged at a speed that removes the cell components denser than the desired cellular component followed by centrifugation at a speed that sediments the desired component. The required protein is extracted from that component by using concentrated salt solutions or , in case of proteins tightly bound to membrane by the use of detrgent solutions or organic solvents such as butanol that solubilizes the lipids. Once a protein has been removed from its natuaral environment , it becomes exposed to many agents that can irreversibly damage it. These influences must be carefully controlled at all stages of a purification process or the yield of the desired protein may be reduced or eliminated. In order to maintain structural integrity of proteins , they are dissolved in buffer solutions effective in pH range over which the proteins are stable. Most proteins except the heat stable proteins are easily denatured by high temperatures so the protein purification is carried out at temperature near 00C. Cells contain proteases which are liberated upon cell lysis . These proteases can destroy the proteins , liberated in the solution. In order to protect the proteins from these proteases, certain chemical agents like PMSF are added in buffer, which inhibit proteases without affecting the target protein.

EXPERIMENT NO. 16 OBJECTIVE: Extraction of total proteins from wheat seeds. PRINCIPLE: Wheat seeds are finally grinded in pestle mortar to obtain a fine powder . Wheat seed protein is solubilized by addition of extraction buffer which contain salts , EDTA. PMSF is added to protect the proteins from proteases. Cellular debris is removed by centrifugation. Proteins come in supernatant. REQUIREMENT: Pestle & mortar, EDTA, NaCl, sodium phosphate, PMSF. MATERIAL & REAGENTS: 1. Extraction buffer (EB) (pH 7.2) EDTA 5 mM NaCl 50 mM Na Phosphate 25 mM 2. PMSF (stock, 0.1M in alcohol) PROTOCOL: 1. 2. 3. 4. 5. 30 g of wheat seeds were finally grinded in pestle mortar with liq. N2. The powder was stirred with 30 ml of cold extraction buffer. Add PMSF to a final concentration of 2 mM. The mixture was centrifuged at 10,000 g for 20 min at 40C. Supernatant was colleted in aliquots and stored at -200C.

OBSERVATION: Weight of seeds (g) Amount of protein/g of sample Remarks

Study Questions: 1. Why is PMSF added in extraction buffer ? 2. What are the functions of NaCl and EDTA in extraction buffer ? 3. What is the prinbciple of salting in ?

EXPERIMENT NO.17 OBJECTIVE: Extraction of total protein from bacterial cells. PRINCIPLE: Bacterial cells are suspended in a buffer containing PMSF, which

inhibits proteases. Cells are broken open by ultrasonication. Sonicated suspension is centrifuged and supernatant containing the protein is collected. REQUIREMENTS: Bacterial culture, NaCl, sonicator, NaH2PO4, Na2HPO4, PMSF Solutions: 1. Normal saline: 0.9% NaCl 2. Phosphate buffer: 0.1M (pH 7.0) PROCEDURE: 1. Bacterial culture was harvested at 10,000 rpm for 5 min. 2. Pellets were washed twice with normal saline 3. Suspend the pellet in 0.1 M phosphate buffer (pH 7.0). (1g pellet in 1ml) 4. Sonicate the bacterial cells by 6 cycles of 30 secs each with 10 sec interval. 5. Add PMSF to the final conc.of 1mM. 6. Sonicated suspension is centrifuged at 10,000 rpm for 10 min at 40C. 7. Supernatant is collected and stored in aliquots at -200C. OBSERVATION : 1. OD600 of bacterial culture used = 2. Weight of bacterial cell mass = 3. Protein concentration (mg/ml ) =

EXPERIMENT NO.18 OBJECTIVE : Extraction of total protein from fungal mycelium. PRINCIPLE : Fungal mycelium is mechanically lysed by grinding in liquid N2 in pestle and mortar. Extraction buffer is added to further lyse fungal cells and solubilize the fungal proteins. Extraction buffer contains ascorbic acid which acts as antioxidant. Beta mercaptoethanol and detergent in less concentration is also added to further solubilize the protein. PMSF acts as protease inhibitor. PVP binds the phenolic compounds. EDTA and EGTA act as chelating agents MATERIAL & REAGENTS : Tris-base, EDTA, EGTA, TritonX-100, mercaptoetahnol (BME), Ascrobic acid, Polyvinyl pyrolidone (PVP), Phenyl Methane sulfonyl fluoride (PMSF). SOLUTIONS: Extraction buffer (pH 7.5) Tris-base EDTA (Na2) EGTA TritonX-100 BME Ascorbic acid PMSF PVP Stock solution of PMSF (1 M) 174 mg/ml in 90% alcohol. PROCEDURE 1. Freeze dry the fungal mycelia. 2. Take about 2g of mycelia (freeze dried) or ~10g of wet mycelia. 3. Cell disruption is done by pestle and mortar in the presence of liq. N 2. Also add extraction buffer during grinding (200 mg dry mycelia/ ml buffer) (1 g wet mycelia/ ml buffer). 4. Centrifuge the lysate at 10-12000 rpm for 30 min at 40C. 5. Collect the supernatant in aliquots and store at -200C. 6. Estimate the total protein in the sample by Bradford method. 50mM 50mM 10mM 0.5% 0.3% 0.39% 2mM 0.4%

OBSERVATION ; Weight of fungal mycelia = Protein concentration in mg/ml = Amount of protein per gm of tissue =

EXPERIMENT NO. 19 OBJECTIVE: Extraction of protein from plant (Brassica) leaves PRINCIPLE: Plant cells are mechanically lysed by grinding the tissue or leaves in mortar and pestle in presence of liquid N2. Leaf protein is thn solubilized in Tris buffer and cellular debris is removed by repeated centrifugation. MATERIAL AND REAGENTS: Mortar and pestle (autoclaved), ice, ice bucket, centrifuge tubes. Extraction buffer (100 ml) 0.2 M Tris.Cl (pH = 8.0). PROTOCOL: 1. Weigh 1 gram of plant leaves. 2. Transfer the leaves in to mortar and add 5 ml of 0.2 M Tris.Cl (pH=8.0). 3. Keep the mortar in ice bucket and crush the leaves with the help of pestle for 20 min. till a fine slurry is made. 4. Transfer the slurry in centrifuge tubes and centrifuge at 10,000 rpm at 40C for 20 min. 5. Transfer the supernatant in fresh tubes. If supernatant has some particulate matter or debris , centrifuge again at 10000rpm for 5 min. Store the supernatant at 40C till further use and quantification. OBSERVATION : Amount of leaf tissue taken = Protein concentration in mg/ml = Protein amount per gm of tissue = QUESTIONS : 1. What are different factors upon which extraction of protein depend upon ? 2. What are the components of Tris buffer and how will you prepare it ? 3. How will you solve the problem of phenolics while extracting protein ?

Pitfalls and trouble shooting :

Problem Improper extraction Cause Crushing of seeds may not be proper Remedy Use fine glass beads or cover slips along with sample while crushing them

QUANTIATION OF PROTEIN CONCENTRION

Review: the Beer-Lambert Law

log(1/T) = cl = A T = 1/10A
Values for and l Path length l is usually in units of cm. (note: most spectrophotometers are designed to accept 1cm wide cuvettes) Molar extinction coefficient has units of M-1 cm-1 and is a constant of proportionality that relates the absorption of molar solutions Mass extinction coefficient 1% refers to the absorbance of a 1% by mass solution. Typically this refers to an aqueous solution that we can take to have a density of 1000g/L. A 1% by mass aqueous solution would therefore refer to the dissolution of 10g/L, or a 10mg/ml solution of the molecule of interest. Since the absorbance of a molecule is a function of the wavelength (i.e. the absorption is not equal for every wavelength) the extinction coefficient must also reference a wavelength. This is typically done using a subscript: = 14.5 g-1 L cm-1 In this case a 10mg/ml solution of the molecule will have an absorbance reading of 14.5 (dimensionless units) at = 280nm (the absorption at other wavelengths may not be known). The units of
280nm

1%

concentration are g/L, thus

will have dimensions of g-1 L cm-1.

Why is it important to be able to quantitate protein concentration in a sample? An important application of "Biotechnology" is the production of proteins as commercial products. Such products might have pharmaceutical applications (e.g. insulin, human growth hormone, tissue plasminogen activator, erythropoietin, blood clotting factor VIII.), industrial applications (e.g. subtilisin (an enzyme in detergents), 2,5-diketo-D-gluconate reductase (an enzyme in vitamin C production), as materials (e.g. silk protein in textiles, barnacle adhesion protein as a glue). In these cases, there are various aspects of successful production that require quantitation: How much of the protein can be produced (i.e. what is the efficiency of production)? How pure is the protein that is produced (industrial applications may require 90% pure, pharmaceutical applications may require 99.999% pure)

Such proteins may be isolated from natural sources (e.g. blood clotting factor VIII may be extracted from human blood), or they may be produced recombinantly (e.g. E. coli bacterial cells can be genetically engineered to produce human growth hormone). In both cases, it may be necessary to purify the protein using a series of fractionation steps. We will go into more detail about such fractionation steps in a later lecture, but the general idea is that a heterogeneous mixture of molecules can be fractionated based upon some physical property of the molecules. The following are properties that can be used to fractionate a heterogeneous mixture of biomolecules: Molecular mass (i.e. "big" molecules can be separated from "small" molecules) pKa (i.e. "acidic" molecules can be separated from "basic" molecules) Hydrophobicity (i.e. non-polar molecules can be separated from polar molecules) For such fractionation steps involving proteins, we need to keep track of how much of the contaminating proteins went into one fraction and how much of our desired protein went into the other fraction. Although the details are somewhat more complicated than this simple description, it is important to be able to quantitate protein concentration to be able to effectively purify a protein of interest. Once a protein is pure, it may be of considerable economic interest to be able to quantify the yield (and, therefore, be able to determine how much it cost to produce a given mass of protein). For example, the only source for human growth hormone (to treat small stature) used to be to extract it from human pituitary glands harvested from the brains of cadavers. Suffice it to say, this made the protein extremely expensive. Furthermore, the isolation from human tissues meant that the sample could also be potentially contaminated with human pathogens (hepatitis, CJD, AIDS, etc.). With the advent of genetic engineering, the production of human growth hormone by bacterial cells (i.e. E. coli) meant that relative large quantities could be produced far cheaper (and with no threat of human pathogens). Why not just weigh the protein? Most samples are typically quantities of milligrams or even micrograms, not grams, and thus, it is difficult to transfer and measure such small amounts Water is present in proteins, and it is extremely difficult to remove all the water (some water molecules hydrogen bond extremely tightly to proteins). Thus, the mass measurement would include some waters, and would increase the apparent mass of the protein Absorbance spectra of biological molecules Proteins

Proteins do not absorb in the visible wavelength unless they have a prosthetic group (e.g. Fe2+) or an unnatural amino acid. However, the amino acids tryptophan, tyrosine and cysteine absorb light in the UV wavelength:

Tryptophan has a peak of absorption at 280nm in the UV range This is a useful wavelength to quantitate the absorption of tryptophan Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for proteins containing Trp)

Nucleic acids The aromatic rings in the bases of nucleic acids also absorb in the UV range:

Each DNA and RNA base has a slightly different absorption spectrum

10

260 or 280nm is a typically useful wavelength to monitor concentration of nucleic acids

Note that samples of nucleic acids and proteins can both absorb at 280nm, therefore, samples of biological molecules should be pure in order to quantitate using UV absorption spectroscopy (any contaminating nucleic acids in a protein sample will increase the apparent absorbance, likewise for contaminating proteins in a nucleic acid sample). Important aspects of quantification of proteins using UV absorbance If a protein contains Trp, Tyr or Cys residues it will absorb in the UV. If it does not contain these amino acids, it will not absorb UV light, and we cannot quantify it using this method Multiple Trp, Tyr or Cys residues will contribute to the Extinction coefficient for the protein. Thus, we need to know how many of these residues are present in the protein to know the correct extinction coefficient Nucleic acids (DNA, RNA) contaminant will also absorb UV light, as will other proteins with Trp, Tyr and Cys residues. Thus, the sample must be PURE to use UV absorption to quantify a protein

Molar extinction coefficients of Trp, Tyr and Cys amino acids: Amino Acid E280nm (M-1 cm-1) Trp 5690 Tyr 1280 Cys 120 Example: Bovine insulin contains 4 Tyr residues, 6 Cys residues and 0 Trp residues. We can determine the expected molar extinction coefficient at 280nm, E280nm, by the following calculation: E280nm = (0)(5690) + (4)(1280) + (6)(120) E280nm = 5840 M-1 cm-1 Thus, a 1.0M solution of pure bovine insulin would give an absorbance of 5,840 at 280nm (obviously, it would have to be diluted considerably to be read accurately). A useful expression relating the parameters of E, concentration (C) and A are derived from the Beer-Lambert law (assuming 1cm path length): A/E = C For example, if a sample of bovine insulin was observed to give an absorbance at 280nm of 0.745 we could calculate the concentration to be: 0.745/5840 M-1 cm-1 = C C = 1.28 x 10-4M (note: cm-1 drops out with 1cm pathlength) It should be noted that a deuterium lamp is required for UV spectrophotometry, as well as quartz cuvettes (since glass absorbs UV light) 11

Colorimetric (chromogenic) methods of protein concentration determination Chromo means color and genesis mean creation, so chromogenic means the "creation of color". Color means color (duh) and metric means to measure, so colorimetric is to "measure color". Both terms refer to the same sort of thing in the present case - we can modify the protein sample with appropriate reagents so as to produce a color reaction (in visible spectrum) and measure protein concentration using a VIS spectrophotometer. Advantages are: Cheap lamp! (tungsten light bulb versus deuterium for UV) Cheap cuvette! (cheap glass or plastic versus quartz) Not contaminating absorbance from proteins or nucleic acids! (no absorption in VIS spectrum) We will consider three methods: The Biuret, Lowry and Bradford methods of colorimetric determination of proteins. Biuret Under high pH (alkaline) conditions the copper II ion (Cu2+) is believed to form a complex with peptide nitrogens of proteins:

This complex absorbs light at 550nm and has the following useful properties: It is dependent upon at least a dipeptide structure (see above), thus, contaminating amino acids will not contribute to the 550nm absorption

12

Lowry

The binding depends upon the peptide backbone nitrogen and not the side chain functional group. Thus, the binding is independent of sequence Nucleic acids do not interfere since they don't share the peptide backbone structure However, ammonia and certain amines can interfere. Thus, ammonium sulfate salts, and the common biological buffer TRIS (tris hydroxymethyl amino ethane) will provide a false positive and cannot be present in the sample Also, the absorbtion is relatively weak, thus, the method is somewhat insensitive and requires a relatively high concentration of protein

The Lowry method is a modification of the Biuret method. After treatment with Copper II, the protein is treated with phosphomolybdotungstate mixed acids (acidic compounds of molybdenum and tungsten ions). These acids are known as the Folin-Ciocalteu (or just Folin) reagent. The Folin reagent is added under alkaline conditions, and the Folin reagent is subsequently reduced by the the Copper ions as well as Tyr, Trp and polar amino acid side chains. The product of this reaction is heteropolymolybdenum blue, which absorbs strongly at 750nm. This assay has the following properties: More sensitive than the Biuret assay (can detect lower concentrations of protein) Somewhat dependent upon amino acid composition (i.e. relative concentrations of Tyr, Trp and polar amino acids) Absobtion reaction is linearly dependent upon protein concentration, but only at low concentrations of protein (i.e. the standard curve and assay must be performed at a low concentration regime). Sensitive to contaminants as with the Biuret method, as well as others related to the Folin reagent and redox reactions. More critical to timing and precision of person doing the assay Bradford A dye known as Coomassie Brilliant Blue was developed by the textile industry. It was noticed to stain skin as well as the textiles. Thus, this dye (which normally absorbs at 465nm) was known to bind to proteins and to absorb strongly at 595nm. The dye forms a wide variety of strong, but non-covalent, interactions including hydrogen bonding donor and acceptor interactions as well as hydrophobic (non-polar) interactions.

13

The method is quite simple: a single step in which the dye is added to the protein solution under acidic conditions, and then the absorbance is read at 595nm. However, the method has the following properties: The response is generally independent of the amino acid composition The assay is sensitive, but somewhat non-linear. Thus, a standard curve must always be performed (using known concentrations of pure protein) The dye stains pretty much everything, including cuvettes, floors, countertops. It can be a real mess if spilled (I know this from personal experience). And since it is a textile dye, if you get it on your clothes, you will need to learn to like blue polka dots.

2004 Michael Blaber

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EXPERIMENT NO. 20
OBJECTIVE: Quantitative estimation of protein by UV absorption spectro photometric method. PRINCIPLE: The absorbance of different molecules occurs at different wavelengths. Molecules can be distinguished from one another by looking at an absorption spectrum. The spectrum is useful because it displays the number of absorption bands that corresponds to each structural group of the molecule. Absorbance is related to the light path and the concentration of the sample by Beers Law, A= abc, where b is the path length, c is the concentration of the absorbing species, and a is the constant of proportionality also known as absorptivity. The absorbance increases as the concentration increases. The instrument used to make spectroscopic measurements is the spectrophotometer. A light source sends beams of light through a monochromator. Next, the incident light (Io) is sent through a cuvette containing the sample of interest. This is represented by I, the light path. Light that is not absorbed by the sample (I) can be detected. This method along with the Bradford dye assay is useful in determining the concentration of proteins. Four spectroscopic methods are routinely used to determine the concentration of protein in a solution. These include measurement of the protein's intrinsic UV absorbance and three methods which generate a protein-dependent color change; the Lowry assay, the Smith copper/bicinchoninic assay and the Bradford dye assay. This investigation uses UV absorbance and the Bradford dye assay. The wavelength at which proteins absorb maximally is 280nm. This absorbance is attributed to aromatic aminoacids such as tryptophan , tyrosine and phenyalanine found in the proteins. This property is utilized in quantification of proteins For this , first a standard curve by plotting known concentration of a standard protein against their absorbance at 280 nm is prepared. Next , the absorbance of unknown protein samples are taken and their concentration is determined by comparing the OD in standard graph. MATERIALS & REAGENTS: 1. Standard protein (BSA) Stock : 1 mg/ml (Weigh 10 mg of BSA and dissolve it in 10 ml of buffer/double distilled water.) 2. Quartz cuvettes PROTOCOL: 1. Prepare different tubes of known concentrations of BSA (10, 30 to 100 g/ml) in extraction buffer/ double distilled water.

15

BSA stock solution 100 l 300 l 500 l 700 l 900 l 1000 l

extraction buffer / double distilled water 900l 700l 500 l 300l 100l 0l

Total volume 1000l 1000l 1000l 1000l 1000l 1000l

2. Take absorbance of above samples at 280 nm against buffer using as blank. 3. Prepare standard calibration curve by plotting known concentration of BSA vs. absorbance at 280 nm. 4. Find out the concentration of protein sample (as isolated in experiment no.1 from this calibration curve. OBSERVATION: BSA concentration (g/ml) O.D. at 280 nm

Unknown protein sample STANDARD CALIBRATION CURVE:

16

OD (280 nm) of protein sample: Concentration of sample as obtained from calibration curve:

Questions: 1. Name the amino acids which contribute to the absorption maxima of protein at 280 nm.

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EXPERIMENT NO. 21 QUANTITATIVE ESTIMATION OF PROTEIN BY BRADFORDS METHOD OBJECTIVE: Quantitative estimation of protein by Bradfords method. PRINCIPLE: The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. The Bradford assay works by the action of Coomassie brilliant blue G-250 dye (CBBG). This dye specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. CBBG binds to these residues in the anionic form, which has an absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red). The assay is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG complex with the protein. Chemical Structure of Coommassie Brilliant Blue G-250

Typical Bradford Assay Standard Curve It should be emphasized that the absorption spectra of the two forms of the dye overlap. This causes the assay to respond non-linearly in the standard curve. The assay does perform linearly over short concentration stretches, and this has most likely resulted in the overall conclusion that the assay is linear. One crucial part of this assay is the buffer blank. Since the assay responds non-linearly it is highly important to lock down the zero point. Because this point is so important to the

18

curve fit, it is highly recommended that at least two buffer blanks be performed.

The choice of standard for this assay is very crucial to the success of the assay. Many investigators have noted abnormalities of using various standards with the Bradford assay. BSA was the original standard of choice. However, it has been noted that BSA has a double than normal response in the assay and may not always be suitable. Several researchers therefore use Imunnoglobulin G (IgG) as the preferred standard for the assay. The CBBG dye used in the assay binds to quartz cuvettes quite strongly. Therefore, glass or plastic cuvettes should be utilized. Since this assay has a general tendency to bind to cuvettes, it is highly recommended to use disposable plastic cuvettes. The Bradford dye assay is based on the equilibrium between three forms of Coomassie Blue G dye. Under strongly acid conditions, the dye is most stable as a doubly-protonated red form. Upon binding to basic and aromatic amino acid residues of proteins,, however, it is most stable as an unprotonated, blue form.

Detection Limitations 1-200 g . Advantages Fast and inexpensive Highly specific for protein Very sensitive 19

Compatible with a wide range of substances Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin) Dye reagent is complex is stable for approximately one hour

Disadvantages Absorbance spectra of the two Coomassie Brilliant Blue G-250 species partially overlap making the standard curve very important Non-linear standard curve over wide ranges Response to different proteins can vary widely, choice of standard is very important MATERIALS AND REAGENTS : Comassie Brilliant Blue (CBB) G-250 , Ethanol, ortho- Phosphoric acid, BSA, double distilled water, Funnel, filter paper , test tubes. SOLUTIONS: 1. Bradford dye: Dissolve 100 mg CBB G-250 in 50 ml ethanol; add 100 ml ortho- phosphoric acid (85% w/v). Make the volume up to 1 lt with double distilled water. Filter the solution and store at 40C in dark colured bottle. The solution prepared should be used within 2 weeks. 2. BSA standard: Stock solution : 1 mg/ml

1. Take 12 glass test tubes having 5, 10, 20, 40, 60, 100 l of BSA in duplicate. Add extraction buffer/ double distilled water to make volume up to 300 l. Prepare the samples and blank as per following table : BSA stock solution Extraction buffer/double Total Volume distilled water ( l) 5 l 295 l 300 l 10 l 290 l 300 l 20 l 280 l 300 l 40 l 260 l 300 l 60 l 240 l 300 l 100 l 200 l 300 l Blank 300 l 300 l Protein sample 1(20 l) 280 l 300 l Protein sample2 (40 l) 260 l 300 l

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3. 4. 5. 6. 7.

Add 3 ml of dye in each tube. Incubate them for 5 min at RT /370C. Now read the absorbance of all samples at 595 nm in colorimeter. Draw a standard graph between the BSA concentration and absorbance. Calculate the amount of protein in the unknown sample with the help of graph.

STANDARD CURVE FOR BSA:

Concentration of protein sample: NOTE: If protein concentration is very low, concentrate the sample by dialysis, as mentioned in next experiment.

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QUESTIONS: 1. Calculate the protein concentration (mg/ml) from following data :

Standard BSA concentration 20 mg/100 ml Total crude extract 3.0 ml Fresh weight of leaves 2.0 grams 4 l of the extract reads O.D. 0.30 Absorbance (O.D.) values for standard are 0.1 ml BSA 0.1 0.2 ml BSA 0.2 0.3 ml BSA 0.3 0.4 ml BSA 0.4 0.5 ml BSA 0.5 2. A unknown protein solution (0.3 ml) is diluted with 0.9 ml water. To 0.5 ml of this diluted solution , biuret reagents were added and colour was developed. It gave OD = 0.12 at 550 nm. In 0.5 ml (4mg/ml) protein solution , biurets reagent was added and colour was developed. It gave OD =0.18 at the same wavelength. Calculate the concentration of protein in (mg/ml) and total protein in unknown solution. 2. What are different methods of quantitation of protein ? 3. Calculate the molecular weight of protein encoded by 5.5 kb of DNA.

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EXPERIMENT NO.22

OBJECTIVE: To concentrate the protein sample by dialysis against sucrose. PRINCIPLE:

Dialysis is the process by which the protein is separated from smaller molecules and ions by enclosing a solution of the impure protein in a partially permeable membrane. Small solute molecules and ions pass out through this while the larger, protein molecules are held back Precipitation and salting out is sometimes used to precipitate out one protein selectively from a solution containing a mixture of different proteins. Adding a very soluble salt (e.g. ammonium sulphate) causes the least soluble protein to precipitate. Proteins have their lowest solubility at their iso-electric point. Selectivity of salting out can be increased by adjusting the pH of the mixture to the isoelectric point of the protein, which is to be precipitated. Organic solvents can also selectively precipitate a particular protein. After an ammonium sulfate precipitation step, or an ion exchange chromatography step, the protein of interest may be in a high salt buffer. This may be undesirable for several reasons. One of the most common methods to get rid of salts is dialysis .The method of dialysis makes use of semi-permeable membranes. This membrane is manufactured in the form of tubing.The main feature of this membrane is that it is porous. However, the pore size is such that while small salt ions can freely pass through the membrane, larger protein molecules cannot (i.e. they are retained). Thus, dialysis membranes are characterized by the molecular mass of the smallest typical globular protein, which it will retain. This is commonly referred to as the cutoff of the tubing (e.g. Spectrapore #6 dialysis tubing has a cutoff of 1,000 Daltons, meaning that a 1,000 Dalton protein will be retained by the tubing but that smaller molecular mass solutes will pass through the tubing) . Prior to dialysis, tubing is boiled in solution of NaHCO3 + EDTA for some time to open the pores of dialysis membrane. Dialysis proceeds by placing a high salt sample in dialysis tubing (i.e. the dialysis "bag") and putting it into the desired low salt buffer. Over time the concentration of low molecular mass solutes within the bag, and in the low salt buffer, will come achieve equilibrium. In practical terms , salt molecules will diffuse out of the bag into the low salt buffer. The buffer volume for the dialysis is a function of the required final concentration of salt in the sample. One consequence of dialysis is that while salt ions are moving out of the bag, water molecules are moving into the bag. Thus the volume of sample may actually increase (the bag will swell) and, therefore, the protein concentration will decrease. Therefore, it is a good idea not to fill the bag completely, but leave a void to allow for potential swelling.

23

Dialysis for concentration of protein sample: If our protein sample is too dilute for our needs, we can concentrate our samples by dialysis. A very simple method is to place our sample in a dialysis bag and place it in beaker filled with sucrose. Water will leave the dialysis bag (it can go through the pores) and hydrate the sucrose. The result is a decrease in volume of buffer in the dialysis bag (the protein will be concentrated). Protein sample thus can be dialyzed to achieve the desired concentration.

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MATERIAL & REAGENTS: Dialysis membrane, sucrose, NaHCO3, EDTA, double distilled water and protein sample. SOLUTIONS : 2% NaHCO3 + 0.05% EDTA (50ml) PROCEDURE : Activation of dialysis membrane 1. Select the dialysis tubing of desired diameter and length. 2. Submerge in a solution containing 2% NaHCO3 and 0.05% EDTA. Boil for 10min. 3. Discard the above solution. Transfer the dialysis tubing to double distilled water and again boil for 10min. This step can be done twice. 4. Cool and place the dialysis tubing in Sodium azide solution (10mM) at 40C to prevent bacterial growth. 5. These activated dialysis tubings can be stored at 40C up to 3 months. B. Concentration of protein sample 1. Seal the dialysis tubing from one end by a clean thread or clip. Check for any leakage. 2. Now pour the protein solution in the tubing. 3. Seal the other end of the tubing also with the thread and again check for the leakage. 4. Place this bag in a beaker containing sucrose powder at 40C, till the volume of the protein sample decrease to the desired volume. 5. Change the sucrose powder after regular intervals. 6. Now take out the protein sample and estimate the protein by Brad fords dye binding method.

OBSERVATION: Concentration of protein sample before Dialysis Concentration of protein sample after dialysis

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Questions : 1. Why activation of dialysis tubing is is needed prior to dialysis? 2. What is the principle behind salting out? 3. How do organic solvents help in protein precipitation?

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EXPERIMENT NO.23 OBJECTIVE: To perform SDS polyacrylamide gel electrophoresis of the protein samples. PRINCIPLE: The separation of macromolecules in an electric field is called electrophoresis. In an early form of electrophoresis, dissolved protein mixtures were placed in a U-shaped bufferfilled channel and subjected to an electric field. Resolution was poor and any disturbance of the apparatus compromised the separation. Gels were developed to serve as solid supports for electrophoresis, so that the separated products remain separated and can be easily stained and handled. The development of the stacking gel, which compresses the sample into bands a few micrometers thick, added a major improvement to the resolution of gels . Other landmark improvements to protein electrophoresis were the use of polyacrylamide for control of separation by molecular size, and the use of sodium dodecyl sulfate (SDS; lauryl sulfate) to denature proteins in order to ensure reproducibility of the technique. SDS is an anionic detergent, meaning its molecules have a net negative charge. It binds to most soluble protein molecules in aqueous solutions over a wide pH range. Polypeptide chains bind amounts of SDS that are proportional to the size of the molecules. The negative charges on SDS destroy most of the complex (secondary and tertiary) structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field.

A polyacrylamide gel with acrylamide content above a critical density restrains larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in molecular weight (MW) of polypeptides. In a gel of uniform density the relative migration distance of a protein (Rf) is negatively proportional to the log of its MW. If proteins of known MW are run simultaneously with the unknowns, the relationship between Rf and MW can be plotted, and the MWs of unknown proteins determined. Protein separation by SDS-PAGE is used to determine the relative abundance of major proteins in a sample, their approximate molecular weights, and in what fractions they can be found. The purity of protein samples can be assessed. Different staining methods can be used to detect rare proteins and to learn something about their biochemical properties. Specialized techniques such as Western blotting, twodimensional electrophoresis, and peptide mapping can be used to detect extremely scarce gene products, to find similarities among them, and to detect and separate isoenzymes ofproteins

27

Gel preparation.

Many systems for protein electrophoresis have been developed, with features such as built in gel casting apparatus and water-cooled jackets. SDS-PAGE can be conducted on pre-cast gels, saving the trouble and hazard of working with acrylamide. Preparations will involve casting of two different layers of acrylamide between glass plates. The lower layer (separating, or resolving, gel) will be responsible for actually separating polypeptides by size. The upper layer (stacking gel) will include the sample wells, and will be of a composition that causes the samples to become compressed (stacked) in order to have thin bands and correspondingly better resolution among bands than if we did not stack them 28

Separating Gel Preparation

In the Laemmli system for SDS-PAGE, proteins are pulled into an upper (stacking) gel, then begin to separate when they reach a lower (separating) gel of different compositiion. A discontinuous gel must therefore be poured in two stages. Acrylamide polymerizes spontaneously in the absence of oxygen, so the polymerization process involves complete removal of oxygen from the solution. From 30%acrylamide stock (see notes below) we prepare gels of compostion 7 to 15% acrylamide, depending on the range of proteins that we wish to separate. Polymerization is initiatiated by adding freshly prepared10% ammonium persulfate (AP) to the mix followed by N-, N'-tetramethylenediamine (TEMED). The amounts of each depend on the quality of acrylamide used, and should be determined in advance by trial and error. Once the catalysts are added, polymerization may occur quickly, thus it is necessary to have the casting stand completely ready and to have the overlay solution ready to go .After swirling to mix, the solution is simply poured into the space occupied by the cassettes. Immediately after pouring the gel mix, it must be overlaid with water-saturated butanol to an additional height of 0.5 cm or so (butanol is the top layer in the stock container). Adding butanol to a single cassette will drive the acrylamide mix down, raising 29

the level in the others, so care must be taken to distribute the butanol equally among the cassettes. The purpose of butanol is to produce a smooth, completely level surface on top of the separating gel, so that bands are straight and uniform. Butanol holds very little water in solution, forming a neat layer on top. Water also makes an effective overlay but there remains possibility of mixing it with the acrylamide solution and diluting it. Polymerization can be confirmed by checking some of the remaining gel mix after 10 min or so. When the remaining gel mix has polymerized, it means the separating gel is set. It should not take more than 15 minutes for any of the gel mixes to polymerize. If it hasn't gelled by that time, something is probably wrong.

Stacking gel preparation

Ten ml of stacking gel mix is sufficient for three cassettes, however for the sake of accuracy it may be preferable to make 20 or 30 ml. Excess can be rinsed and tossed into a wastebasket after it polymerizes. Our stacking gel buffer stock consists of 0.5 M Tris-Cl, pH 6.8, with 0.4% SDS. Typical stackers are 3 to 5% acrylamide. We use 5% in order to permit stacking of very large proteins and still retain sufficient mechanical strength to make good sample wells. Before adding the final two components, which will start polymerization, the butanol or water should be poured off the separating gels into a sink with tap water running and excess butanol/acrylamide removed from the surfaces with a pipet. We use AP and TEMED in similar proportions as for the separating gel mix, although we sometimes increase the amount of one or both components since lower percentage acrylamide solutions tend to polymerize more slowly. After adding AP and TEMED we immediately swirl the mix and pour it into the cassettes to the tops of the plates. We insert combs one at a time, taking care not to catch bubbles under the teeth, and adjust to make them even if necessary, scraping excess stacking mix off later.

Notes on gel preparation

Acrylamide is a toxic substance so use care and wear gloves while handling solutions that contain it. Use in a well ventilated area, and report any spills. Stock solutions should be kept in a fume hood. An erlenmeyer flask is best for mixing acrylamide, since the narrow neck can be stoppered to prevent toxic fumes from escaping. The wide bottom allows for a large surface area, so that oxygen can be quickly removed from the solution when it is placed under a vacuum. Acrylamide gel stock is labeled according to acrylamide monomer content. This formulation uses an acrylamide stock of 29.2% acrylamide and 0.8% bisacrylamide, the cross-linker (cross linking gives the gel its mechanical stability). The stock solution is labeled 30% T (29.2 + 0.8 = 30), 2.5% Cbis (0.8 is 2.5% of 30). Depending on the properties of the lots of ammonium persulfate, TEMED, and acrylamide, the exact volumes of the catalysts may have to be adjusted to achieve desired polymerization times. 30

A separating gel of given acrylamide concentration separates proteins effectively within a characteristic range. The largest polypeptides can enter a low percentage gel readily, and are fairly well separated. However, such a gel has a relatively low cutoff. That is, polypeptides below a particluar size are not restricted at all by the gel, and all move at the same pace, along with the tracking dye, regardless of size. A gel of 7% acrylamide composition typically has a cutoff of 45 kiloDaltons. A gel of very high percentage acrylamide may restrict all of the proteins in a mixture. The smallest protein of any significance among the fractions of mammalian blood is hemoglobin (14 kD). The hemoglobin band is readily resolved in a 15% acrylamide gel, but is buried in the dye front in a 7% gel. The problem with running just a 15% gel is that the heavier proteins are so restricted that they are jammed near the top of the gel and are not easily resolved from one another. In fact, it is a good idea to forget about analyzing the top third of such a gel. To take advantage of the characteristics of both low and high percentage gels, we usually run both.

Assembly, Loading, and Running of Gels

The assembly of a gel running stand varies with the type of apparatus. The top of the cassette must be continuous with an upper buffer chamber and the bottom must be continuous with a lower chamber so that current will run through the gel itself. The cassette must be sealed in place using gaskets or a sealant such as agarose. We fill both the upper and lower buffer compartments with an electrode buffer (running buffer) consisting of 25 mM Tris, 192 mM glycine, 0.1% sodium dodecyl sulfate. We remove the comb from the gel before filling the upper buffer compartment. Hamilton syringes work well for loading samples into the wells. Ideally, the glycerol in a sample causes it to sink neatly to the bottom of the well, allowing 20-100 ul of sample to be loaded. The anode (+ electrode) must be connected to the bottom chamber and the cathode to the top chamber. The negatively-charged proteins will move toward the anode, of course. Gels are usually run at a voltage that will run the tracking dye to the bottom as quickly as possible without overheating the gels. Overheating can distort the acrylamide or even crack the plates. The voltage to be used is determined empirically . When the dye front is nearly at the bottom of the gel it is time to stop the run. For low percent gels with a tight dye front, the dye should be on the verge of running off the gel. When the percent acrylamide is high the dye front may be diffuse, since the dye is not homogeneous. If you know the approximate position of the lowest protein band you can let the dye run off. Before removing gels the power must be turned off and cables removed (using one hand, to avoid making a circuit). For removal of the gel from the cassette The plates are separated and the gel is dropped into a staining dish containing deionized water. After a quick rinse, the water is poured off and stain added. Staining usually requires incubation overnight, with agitation.

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Staining protein gels

A commonly used stain for detecting proteins in polyacrylamide gels is 0.1% Coomassie Blue dye in 50% methanol, 10%glacial acetic acid. Acidified methanol precipitates the proteins. Staining is usually done overnight with agitation. The agitation circulates the dye, facilitating penetration, and helps ensure uniformity of staining. The dye actually penetrates the entire gel, however it only sticks permanently to the proteins. Excess dye is washed out by 'destaining' with acetic acid/methanol, also with agitation. It is most efficient to destain in two steps, starting with 50% methanol, 10% acetic acid for 1-2 hours, then using 7% methanol, 10% acetic methanol to finish. The first solution shrinks the gel, squeezing out much of the liquid component, and the gel swells and clears in the second solution. Properly stained/destained gels should display a pattern of blue protein bands against a clear background. The gels can be dried down or photographed for later analysis and documentation. The original dye front, consisting of bromphenol blue dye, disappears during the process. In fact, bromphenol blue is a pH indicator which turns light yellow under acid conditions, prior to being washed out. In low percentage gels, sufficient protein may run with the dye front so that the position of the bromphenol blue front is permanently marked with unresolved proteins, often forming a continuous "front" across the bottom of the gel. In higher % gels, a distinct dye front is usually not obtained. Coomassie blue may not stain some proteins, especially those with high carbohydrate content. Stains such as periodic acid-Schiff (PAS), fast green, or Kodak 'Stain's all' may detect different patterns. Silver staining is generally used when detection of very faint proteins is necessary.

Mobility
Mobility = (applied voltage) (net charge on molecule)/(friction on molecule) This equation reduces to: mobility = 1/MW when using SDS-PAGE. SDS is an anionic detergent which denatures protein by "wrapping around" the polypeptide backbone.SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In doing so, SDS confers a negative charge to the polypeptide in proportion to its length. EXCEPTIONS: highly basic or acidic proteins, very small proteins, membrane, glycosylated or phosphorylated proteins. These extremes often contribute to variability in the apparent molecular weight of a protein. It is usually necessary to use dithiothreitol (DTT) or 2-mercaptoethanol as a reducing agent to disrupt sulfhydral bonds between adjacent cysteine residues. All proteins are contained in the same gel slab within the

32

same electrophoresis chamber, and therefore the applied voltage experienced is the same for all proteins Friction. The factors contributing to friction are the size and shape of the protein. Since SDS/DTT neutralizes the shape, proteins are thereby separated on the basis of SIZE (MW). Understanding the relationship of mobility and size allows you to determine what percentage of acrylamide best suits your resolution needs MATERIALS: Acrylamide, bis- acrylamide, SDS, Tris-HCl, Tris-base, Glycin, mercaptoethanol (BME), Bromophenol blue. Glycerol, Comassie brilliant blue R-250, TEMED, Ammonium per sulphate (APS), EDTA, glacial acetic acid and methanol. SOLUTIONS: 1. 30% Acrylamide & bis-acrylamide stock solution: 29.2 g of acrylamide and 0.8 g of N, N- methylene bis acrylamide dissolved in 100 ml of ddw. Stir at magnetic stirrer and warm it slightly so as to dissolve it completely. Filter and Store at 40C in a dark colured bottle. 2. 10 % SDS : 10 g SDS was dissolved in 100 ml of double distilled water. Keep at 37 0C or warm it slightly for complete dissolution. 3. Stacking gel buffer: 0.5 M Tris-HCl (pH 6.8). 4. Separating gel buffer: 1.5 M Tris-HCl (pH 8.8). 5. Electrophoresis buffer: (pH 8.3) (Tris-glycine buffer) Tris base 25 mM Glycine 192 mM SDS 0.1% 6. Sample buffer(2X) SDS-PAGE Tris-HCl (pH 6.8) SDS Glycerol BME EDTA BPB 7. Fixing solution 50% Methanol 10% glacial acetic acid 8. Staining solution 50% methanol 62 mM 2% 10% 5% 0.1% 0.1% Native PAGE Same Not added Same Not added Same Same

33

10% glacial acetic acid 0.1% CBB - R250 9. Destaining solution 10% methanol 7% glacial acetic acid 10. 10 % Ammonium per sulphate : 0.1g/ml Procedure 1. Composition of 10% separating gel (20ml) 30% Acrylamide stock : 8.0ml Separating gel buffer : 5.0ml 10% SDS : 0.1ml 10% APS : 0.2ml TEMED : 10 l ddw : 6.69ml 2. Composition of 5% stacking gel (8ml) 30% Acrylamide stock : 1.3ml Stacking gel buffer : 2.0ml 10% SDS : 80 l 10% APS : 80 l TEMED : 10 l Ddw : 4.53ml APS and TEMED are added just before pouring of the gel solution as TEMED initiates the cross-linking of the gel. For native PAGE the composition of stacking and separating gel will be the same except the SDS. There will be no SDS in the solutions of native- PAGE. 3. Carefully clean the glass plates with warm soapy water. Rinse them thoroughly with ddw. Dry and wipe them with ethanol. 4. Assemble the gel plates with the spacers and the clamp. Now pour the separating gel solution prepared, between the glass plates. Overlay it with water and allow it to polymerize for 30 40 min at 370C. 5. Remove the overlay solution. Wash the gel 2-3 times with ddw. Now pour 5% stacking gel prepared. Insert the comb and let the gel again polymerize. 6. Remove the comb, clean the wells with a syringe and assemble the gel plates in the electrophoresis chamber, filled with the electrophoresis buffer. Pre run it at 80V for 15min. Sample preparation: 1. Approx 50 g of protein sample was taken with 2x sample buffer in 1:1 ratio. This was boiled in boiling water bath for 5 min. For native PAGE sample are not boiled 2. Centrifuge the samples at 10,000 rpm for 10 min. 3. Load the samples with the help of micropipette. 4. Run the gel at 2-3 mA/lane at constant voltage of 80Volts till the sample is in slacking gel and after that the voltage was raised to 100 volts. Allow the gel to run till the dye reach 0.5 cm from the lower edge of the gel.

34

5.

After completion of electrophoresis gel was taken out in a tray containing the fixing solution. The gel was left in it for 30 min. 6. Leave the gel in staining solution O/N. 7. Next day gel is taken out and kept in destaining solution. 2-3 changes of destain are given. The gel is destained till the bands are clear.

OBSERVATION TABLE : Well no. Amount protein of Sample buffer Double Distilled water Total volume loaded

SAMPLE GEL

PASTE HERE YOUR GEL PHOTOGRAPH

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TROUBLE SHOOTINGS
Smeared Protein Gels
Example 1
Smearing can have a variety of causes, but most commonly it is due to an unevenly poured acrylamide mixture or due to gross overloading of protein.

In this example the gel was not properly poured, so that the lower half had begun to polymerize before the upper part was poured. The first gel mix began to polymerize too quickly. Rather than prepare a new gel cassette, the students simply stopped pouring, prepared a new mix, and poured it on top of the old. Obviously, the bond wasn't particularly good.

Example 2

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In this example a lot of protein was loaded in each of the wells, all the way across. Most of the lanes can still be interpreted, but smearing is particularly evident in lanes 3 and 4. Those lanes contained primarily one protein (hemoglobin subunit), as did lanes 9 and 10. However, the group that loaded 3 and 4 underestimated the protein concentrations in their red cell lysate and red cell cytoplasm fractions. Those fractions contained so much protein that samples had to be diluted prior to preparing a protein assay tube. The students forgot to take the dilution factor into consideration when determining protein concentration. The capacity of a mini-gel for a mixed protein sample is 20 to 40 micrograms/well, depending on the resolution needed and number individual polypeptides in the mix. However, if a pure protein is loaded, one single band will contain all 20-40 micrograms, and the result is a mess.

Example 3
Here is another example of gross overloading, and it appears these students made the same mistake as in example 2. It looks as though there was some inconsistency in the gel also.

Example 4
Smearing can be a normal consequence of running membrane-associated proteins with a high lipid content in a discontinuous gel. In discontinuous PAGE, sample proteins are super-concentrated by a combination of two factors. First, the rate of migration is suddenly slowed as the sample moves from a non-restrictive stacking gel to a separating gel that restricts movement. Second (and more important), a pH change affects the electrophoretic mobility of proteins in the sample, causing the sample to check up abruptly. A sample of a half cm or so in height becomes compressed into a layer of bands a few micrometers thick, and the local concentration of protein goes up considerably. Membrane-associated proteins tend to precipate at lower concentrations, thus lanes containing such proteins often have a dark band of precipitated proteins at the very top. As electrophoresis proceeds the precipates re-dissolve and enter the gel continuously, thus forming a continuous dark background of unresolved polypeptides. Many of the membrane samples shown on these gels will show that characteristic.

37

The resolved bands in the membrane protein lanes (4 and 7) appear to consist of similar amounts of protein, however in lane 4 the capacity of the gel for at least some of the membrane proteins was exceeded, resulting in a dark smear.

Streaked Protein Gels

Example 1
This one isn't so bad. The lane with the streaks (left of lane 5) is quite usable, in fact. The streaks came about because a portion of the more concentrated proteins in the membrane fraction precipated during the stacking process, then returned to solution after a fairly short time. Because of slight imperfections in the separating gel surface (it may have been allowed to dry a little before adding the stacking gel), the protein concentration was not uniform across the lane, and streaks rather than a uniform darker background are the result.

The electric field was quite uniform, and diffusion minimal, otherwise the streaks would not remain as narrow vertical lines.

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Example 2
The fourth lane is streaked due to the effect described above. The electrical field was affected by the non-uniform concentration in the fourth lane (the dark one with streaks),, causing a narrowing of the bands.

Note that the dye front was lost in this gel - it was allowed to run too long.

Example 3
This one kind of stinks. Evidently, this group did not use an overlay after pouring the first gel layer. The result was a separating gel with the very rough top that you see here. As samples were stacked they were disproportionately concentrated in the 'valleys,' where the proteins precipitated. The precipitates re-dissolved gradually during the run, forming streaks instead of resolving into discrete bands.

Bands Too Light

Example 1
It looks like this problem was caused by poorly made wells and/or careless loading of sample onto the gel. The background isdark and uneven, suggesting that protein was in the running buffer itself. Most likely the apparatus was jarred and much of the samples was slopped out of the wells. The spilled sample would not resolve into bands, since it continuously entered the gel from the upper buffer compartment.

Note the presence of a horizontal line, continuous along several lanes (arrow). That would suggest that the wells were too shallow, and some material spilled into adjacent wells.

39

This symptom also shows up in perfectly good gels when the electrodes are hooked up backwards for a few minutes. Protein migrates out of the wells instead of into the stacking gel. Catching the problem within a few minutes salvages some of the information, but the pH effect that causes efficient stacking is compromised, sample is lost, and protein contaminates the running buffer.

Example 2
Judging from the lack of other symptoms, it appears that the gel was simply poorly stained. Coomassie blue dye staining solution can become contaminated with SDS if it is recycled. The dye becomes less effective and proteins don't show up as well as with fresh dye. As long as the proteins were precipitated in the gel by the acidified alcohol in the stain solution, the problem could be corrected simply by re-staining with fresh dye.

Example 3
The 'X' marks the lane next to lane 5 where the amount of protein was overestimated in the original sample, the sample was prepared to the wrong concentration for electrophoresis, or too little was loaded - perhaps sample wasn't properly drawn up into the syringe. The absence of any major bands, compared to the other samples, suggests that the well was simply underloaded.

Example 4
This is one of the more disappointing symptoms you might encounter. Let's say you were extremely meticulous, did everything perfectly, and ran your gel so that the dye front was 40

perfectly even and right at the bottom. You rinsed the gel in deionized water, then left it on the shaker. Being late in the day, you forgot to replace the water with the stain. It was stained the next day, but here's the result:

Notice that the smaller proteins diffused rather quickly out of the gel, while the larger ones at the top diffused much more slowly, and took the stain. The acidified alcohol in the stain solution and in the destain solutions is essential, as it precipates proteins, preventing them from diffusing out of the acrylamide matrix.

Missing Dye Front

Example 1 - partial dye front
After doing so much work getting the gel set up, it is a shame to be late taking it down. Relative mobilities cannot be determined with accuracy if the dye front is partially off of the gel.

In this case the gel was internally calibrated - that is, molecular weight standards were included on the gel. An arbitrary reference point can be used for setting up the standard curve, since the lanes were quite even.

41

Example 2 - no dye front

This one was allowed to run until the dye front completely ran off. Since the lanes are somewhat distorted, relative mobility measurement will be compromised. With an intact dye front, lanes could be compared even if the gel was distorted or of different height on one side or the other. Relative migration is the same for a gel of uniform composition, no matter what the actual height of the resolving gel.

Example 3 - no dye front

The curvature of this gel suggests that the spacers on the sides were a bit loose, causing a field effect near the edges (a 'frowning' gel). It will be difficult to get accurate relative mobility measurements because the dye front was allowed to run off. With an intact dye front as a reference point, lanes could be compared with one another despite the 'frown.

Bands Crowded at the Top of the Gel

Example 1 - stopped too soon
It is obvious where the dye front was on this gel. There was room to continue electrophoresis, which would have spread out the bands even more and improved resolution. Note that the pattern is consistent with the other gels that "went the distance." Relative mobility, for gels of identical composition, is the same for identical proteins. The resolution is improved with the distance the proteins are permitted to migrate, until resolution becomes limited by diffusion.

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Example 2 - stopped too soon

This one was looking good - then someone became impatient and stopped the run early. It is usable, but the resolution would have been much improved by allowing the dye front to reach the bottom of the gel. This will happen when you run late, the instructor gets hungry, and you aren't there to keep him from taking down your gel.

Failed Gels - Crooked, Broken, Smeared, Weird

Crooked bands
Examples of this problem are scattered throughout the 'hall of shame.' The surface of the resolving gel was uneven, so that when the samples were stacked, the bands started out with distorted shapes.

Crooked bands also result from an uneven electric field, but usually that type of distortion is symmetrical.

Bad wells
If samples run over into adjacent wells, you will see what looks like a continuous protein band running across the wells (arrow). In fact, that's exactly what it is. This is usually

43

what happens when the wells are too short, or perhaps sample is accidentally spilled outside the wells. In addition, we may have used old sample buffer. An advantage of using Cleland's reagent (dithithreitol, or DTT) to reduce disulfide bonds is that it doesn't stink as badly as 2-mercaptoethanol. However, the latter compound is much more stable. Disulfide bonds link individual polypeptides together and maintain some proteins in a folded state under otherwise denaturing conditions. This causes them to run unpredictably.

Dense Gel
...or should it be dense students? The gel mix was clearly incorrect. The acrylamide concentration was so high that only the smallest proteins entered the gel to any extent. The bands were distorted due to overloading and the fact that beyond a reasonable concentration the most concentrated gels tend not to polymerize evenly.

Broken Gel
This can be really frustrating. You did a very nice job, then the gel broke during handling. Keep in mind that the gel will still be usable as long as you save the pieces.

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Field Effects
To some extent this is normal, although for some reason the field effect was exaggerated here. The spacers interrupt the electric field at the edges of the gel, so that the 'pull' (or 'push' if you prefer) is biased to one side the closer the sample is to the edge of the gel. The result is the 'frowning'effect you see here.

The individual bands sometimes smile, but if the gels have any expression at all, they always frown. I guess you would too if we zapped you with electricity and drowned you in blue dye.

Failed Gels with Multiple Symptoms

Example 1
Many times the gels you run will have a variety of symptoms. Since the object of a critique is to improve your work, it is important to determine what caused the undesirable effects. Then you can take steps to improve the quality of your gels.

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This one has no dye front - electrophoresis was terminated too late. Bands are crooked the top of the resolving gel was uneven, most likely due to failure to use a properly overlay. On the right side, samples were overloaded.

Example 2
This gel shows some field effects (curvature - lanes are not straight). There is some overloading, and some bands are crooked due to an uneven resolving gel surface.

Example 3
Uneven loading (different amounts in adjacent wells) has caused some compression in the standards lane. There are tears in the gel and discoloration throughout. This indicates that it was not properly handled - handling a gel with bare hands leaves protein on the surface that stains with Coomassie. Sometimes you get very nice fingerprints.

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STUDY QUESTIONS 1. Whats the role of TEMED and APS in preparation of stacking gel and separating gel mixture ? 2. Why is glycerol/sucrose is added in sample buffer ? 3. Why is mercaptoethanol added in sample buffer ? 4. Differentiate between Native PAGE and SDS PAGE . 5. A protein mixture needs to be separated on the basis of size only. Which type of PAGE will be done in this case? 6. 7. Why the pH of running buffer and separating gel buffer is kept near to 9.0 ? Why the pH of stacking gel buffer is kept almost 2 units lower than the separating gel buffer ?

8. Whats the difference between Comassie brilliant blue G-250 and R-250? 9. Upon electrophoretic separation of a protein mixture on PAGE , streaking was observed after staining of acrylamide gel. What could be possible causes ? 10. Fill in the blanks: i. Dye used to stain protein gel is______________ ii. Dye used in Bradford reagent is_____________ iii. PMSF is soluble in_______________ iv. ________ and______ help in polymerization of acrylamide gel. 11. PAGE can be used for DNA separation or not ? Discuss. 12. An oligomeric protein with five subunits ( & = 20 kDa , & = 15 kDa and = 12 kDa) is analyzed by both by Native PAGE and SDS PAGE. What pattern will you expect in two cases. 13. An acrylamide separating gel mixture was prepared and poured in between two glass plates for polymerization. The polymerization of separating gel did not occur even after 6 hours. What could be the possible cause?

47

EXPERIMENT NO.24. ELISA

OBJECTIVE: To check the titre of the sera raised against the IgG (calf) and glutenin (wheat) by performing . PRINCIPLE: An antibody reacts with the concerned antigen in a highly specific manner ( i.e. an antibody reacts only with that determinant or region of an antigen for which it is specific ) to produce an antigen-antibody complex. This property can be utilized to either quantitate a specific antigen or specific antibody against a particular antigen. ELISA is enzyme linked immunosorbent assay. The basic principle of ELISA is as follows : The antigen of interest is immobilized on the surface of a microtiter plate ( specially constructed ELISA plates which are commonly used for convenient handling of large numbers of test samples; these plates have small wells at uniform intervals). ELISA plate is a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.

ELISA PLATE Now, antibody specific to the antigen is added and allowed to react with the adsorbed antigen. Unreacted molecules of the antibody are washed away leaving only the antigen antibody complex. The antigen specific antibody is unlabelled. This constitutes the primary reaction and the unlabelled antibodies are therefore known as primary antibodies. After that , secondary antibodies which are anti-antibodies is added in to the wells of microtitre plate. Secondar antibodies remain labeled with an appropriate enzyme molecule in such a way that its anti-antibody activity is not abolished. The unbound secondary antibody is washed away.Then the substrate of enzyme is added along with the necessary reagents to develop a colour. The intensuity of colored developed is an indicator of secondary antibody bound to primary antibody which is in turn an indicator of the quantity of antigen. Therefore intensity of colour is used to determine the quantity of antigen present in a sample . The intensity of colour is read spectorophotometrically by using a computerized ELISA reader.The sensitivity of ELISA is in nanograms ( 10-9g/ml). This method of ELISA is often called as direct antigen coating (DAC) ELISA. This is useful in estimating the amount of specific antibody present in the antiserum used for the primary reaction.

48

Briefly the procedure of DAC ELISA can be summarized diagrammatically as follows :

Coating of antigen on to an ELISA plate

addition of primary antibodies

addition of secondary antibodies labeled with an enzyme

addition of substrate for the enzyme

Positive ELISA Colour developed

Negative ELISA No Colour developed

In another approach, unlabelled primary antibodies specific to antigen of interest are first immobilized on to surface of wells of microtiter plate. The antigen is added and allowed to react with primary antibodies. Next , secondary antibodies which are labeled with an enzyme are added and unreacted antibodies are washed away , substrate of enzyme is added and the intensity of colour is measured. This procedure is known as double antibody sandwitch (DAS) ELISA. It is essential for DAS ELISA that antigen must have atleast two accessible epitopes: one for primary antibody and second for secondary antibody.

49

The enzymes used for labeling of secondary antibodies are horseradish peroxidase, alkaline phosphatase , Beta-galactosidase , lactoperoxidase etc. In case of the peroxidase the substrate hydrogen peroxide (H2O2) is converted in to water and O2 in the presence of electron donors like diaminobenzidine or 4-chloronaphthol, which are themselves oxidized in the reaction. Oxidation of diaminobenzidine produces dark brown colour , while that of 4-chloronaphthol produces purple colour , which is the basis of ELISA. In case of alkaline phosphatase , the substrate para nitrophenyl phosphate (colourless) is cleaved under alkaline condition to produce paranitrophenol which is yellow in colour.Intensity of yellow colour is read at 405 nm. ELISA is already being used in field of medicine to detect serum level or in diagnosis of diseases like AIDS. In developed countries it is also used in diagnosis of several plant diseases. MATERIAL REQUIREMENTS: NaCl, KCl, KH2PO4, Na2HPO4.2H2O. Distilled water, Na2CO3, NaHCO3, BSA Diethanolamine, MgCl2.6H2O, Na Salt of para nitro phenyl phosphate, NaOH, 96-well plates, ELISA reader. SOLUTION: 1. 0.15M Phosphate buffered saline (PBS) (pH 7.4), 1000ml NaCl 8.0 g KCl 0.2 g KH2PO4 0.2 g Na2HPO4.2H2O 1.42 g Adjust the pH 7.4 and make the vol. up to 1000ml with ddw and autoclave. 2. Coating buffer (0.05M Sodium carbonate buffer, pH 9.5) Na2CO3 NaHCO3 1.59 g 2.93 g

3. Blocking solution (2% BSA), 100ml. Dissolve 2g BSA in 100 ml of PBS. 4. Antibody dilution buffer (pH 7.0), 100 ml Dissolve 250 mg of BSA in 100 ml of PBS. Store at 40C. 5. Substrate buffer (pH 9.8), 100 ml Diethanolomine 9.7 ml MgCl2.6H2O 0.1 g

50

Make the volume upto 100 ml with ddw. Store at 40C. 6. Substrate solution (1mg/ml), 10ml Dissolve 10 mg of sodium salt of p-nitrophenyl phosphate in 10ml of substrate buffer. Store at 40C in dark vials. 7. Stop solution (6% or 1.5 M NaOH) Dissolve 6g of NaOH in 100ml of ddw. Make the solution in plastic bottle and store at RT. PROCEDURE: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Coat microtitre plates with different concentrations of proteins in coating buffers keeping the volume constanti.e. 100 l per well of soluble antigen of glutenin and calf IgG. Incubate the plates for 1h at RT. Keep O/N at 40C. Wash the wells with PBS. Fill the wells with blocking solution for 2h at RT (to bind the nonspecific sites) Remove this solution and wash the wells with antibody dilution buffer. 100 l of primary Ab dilutions are done and added. Incuabte for 2h at RT. Wash the wells thrice with antibody dilution buffer. Add 100 l of 1:1000 times diluted alkaline phosphatase conjugated secondary antibody. Incubate for 2h at RT. Wash the plate with antibody dilution buffer 3 times. Add 100 l substrate solution in each well. Incubate the plates in dark for 30 min. Stop the reaction by adding 100 l of 1.5 M NaOH solution (%) Read the plate at 405nm in an ELISA reader. OBSERVATION: Table: Record OD405 values of different sample concentrations. Sample concentration ( g/ml) OD405 value

Make a curve between OD 405 values and different sample concentrations : Determine the concentration of unknown sample if any.

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ELISA TROUBLESHOOTING
PROBLEM : No signal or a very weak signal

Test/find higher affinity primary antibody to use in the system. Are there any compounds in the sample which can interferre with coating of antigen/antibody to the ELISA plate? (like high concentration of urea) Has a key reagent been added? Have some steps of the whole procedure been omitted by mistake? Has a substrate for the enzyme been prepared correctly? Was it a right substrate for the right enzyme? Could a detergent concentration in the wash buffer be too high? They are usually used at 0.01-0.1 %. Remove or decrease detergent concentration in the wash buffer. Do you have any enzyme inhibitor present in your system? Sodium azide will inhibit peroxidase activity. Is conjugate or substrate still active? Test their activity in another system. Check expiry date of the products. Has incubation time with the reagents been correct? Check with the manufacture recommendations. Was the incubation temperature correct? Check if the system was not overheated (>37C) or too cold (room temperature). Was the substrate volume correct? Check your pipet.

PROBLEM: High background present

Non-specific binding of antibodies possible. Modify your blocking conditions.Do not apply extensive washes, since they might contribute to incresed variation by denaturation. Plate not washed enough. Check that all the wells are actually filled with buffer during a washing step. Conjugate concentration was too high. Check with the recommendations of the antibody manufacture. Consider cross-reactivity of the detection antibody with coating antibody. Make a control of just coating antibody/antigen and detection antibody with no sample in between. Too high temperature during whole procedure (>37C). Albumins can in some cases contribute to increased variability by causing separation of already bound proteins from medium binding surfaces.

PROBLEM: Non-specific color development on the plate

Usually results from the not complete washing of the plate. If you titrate your samples, and wash plates manually during the whole procedure, you can start the wash from the wells with the lowest dilution of your samples.

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PROBLEM : Strange results

Check that ELISA plate used was the right one for your application (there are different types of plates available). Check if all the reagent were prepared correctly added in the right sequence. Analyse the whole procedure with the help of the points above.

PROBLEM : Poor standard curve obtained

Washing problem. Wells were not completely aspirated during wash. Plates were stuck on each other during incubations. Keep them separately if they are not rotated. Dilution error. Check your pipets and pipeting technique. Variable absorption of reagents to the plate. Check pH of your coating buffer, which is usually around pH 7.4 if using PBS, or pH 9.6 if using carbonatebicarbonate buffer. Consider using another type of ELISA plates.

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QUESTIONS:
1.What is the ELISA test intended to measure? 2. What would happen if serum were omitted from the ELISA, but all other steps remained the same and were performed properly? 3. What would happen if the anti-human Ig-conjugate were not washed free of the well before the substrate was added?

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EXERCISE 25: QUANTITATIVE PRECIPITIN ASSAY (QPA) OBJECT : Estimation of concentration of polyclonal antibodies in biological fluids by quantitative precipitin assay.
PRINCIPLE & THEORY : Polyclonal antibodies are obtained from immunized animals. These antibodies are used either directly or after processing to certain level of purity. In order to use these antibodies in immunological techniques it is essential to know the exact concentration of the active antibodies. QPA is simple and reliable technique commonly used for estimating active antibody concentration in biological fluids. We shall use a kit which is developed for this purpose and is based on Quantitative Precipitin Assay (QPA) technique and use of UV absorption method for protein estimation. The principle of quantitative precipitin assay is as follows: Addition of a polyvalent antibody to polyvalent antigen results in antigen antibody complex larger enough to precipitate out of solution. The precipitate is produced by the origination of structure called lattice which then precipitates out. The degree of precipitation varies with the ratio of antigen to antibody. When a constant amount of antibody is taken and it is mixed with increasing amounts of antigen, the precipitation of antigen antibody complex increases to a maximum and then decreases. Maximal precipitation occurs when all the antigen and all the antibody are incorporated into a lattice i.e. when antibody and antigen concentrations reach the zone of equivalence. In zone of equivalence both antigen and antibody are in equal concentration. When either antigen or antibody is in excess, smaller number of antigen antibody lattices form, resulting in decreased precipitation. The amounts of maximum precipitate obtained and antigen added in the tube is identified. The difference in these two amounts is the amount of antibody in the tube. MATERIALS : 1. Assay buffer: Dilute 10 X concentrated buffer 10 times with distilled water to get 1X assay buffer on the day of use. 2. Antigen (Ag): Add 2.5 ml of 1X assay buffer in one of the antigen vial. Dissolve the content completely and store at 4-80C (stable for 2 weeks).

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3. Antiserum (Ab): Reconstitute one of the antiserum vial with 2 ml of 1X assay buffer and store at 4-80C (stable for 1 month). 4. 0.2 N NaOH: Dilute 2X concentration NaOH with equal amount of distilled water to get 1X conc. on the day of use.

PROCEDURE
1. Add antigen 1.0 mg/ml, 1X assay buffer and test antiserum/ antibody referring to the following table Tube no. P-1 P-2 P-3 P-4 P-5 P-6 Ag (1.0 mg/ml) in l 0 25 (25 g) 50 (50 g) 75 (75 g) 100 (100 g) 150 (150 g) 1X assay buffer l 250 225 250 175 150 100 Test sera in l 100 100 100 100 100 100

2. Mix and incubate at 370C for 40 min followed by 4-80C for 20 min. Mix gently every 10 min (alternatively it can be incubated O/N in fridge). 3. Centrifuge the tubes for 10-15 min to get a tight pellet. 4. Aspirate the supernatant carefully (remove as much of the supernatant as possible without disturbing the pellet). 5. Resuspend the pellet with approx. 1 ml 1X assay buffer. 6. Centrifuge the tubes for 10-15 min. 7. Aspirate the supernatant carefully. 8. Dissolve the precipitate in 1 to 2 ml of 0.1 N NaOH (1X conc.) 9. Read OD280 OBSERVATION : Samples/tube no. Amount of NAOH Added (M) OD at 280nm Protein content in tube

Calculate the protein content in different samples by following formula:

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Protein content in the precipitate A280 M = mg 1.4 Where M is the amount of NaOH added. Calculate Amount of antibody = amount of precipitate antigen added.

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EXERCISE NO.26 OBJECTIVES : Immunization of rabbit with protein extracted from wheat and serum (Glutenin and globulin) PRINCIPLE & THEORY : Production of antiserum which contain antibodies against a particular antigen is important step for various molecular biological procedures like western blotting. Immunization is a procedure by which an antigen is injected in to an animal for the purpose of raising antibodies. An antigen is actually an immunogen which invokes an immune response when injected in an animal. Several factors are considered while performing immunization: 1. Selection of the animal : It is better to use a syngenic animal whose genetic constitution is known. The animal selected for immunization should be such that in which antigen injected is foreign. E.g. Better animal raising antibodies against BSA would be rabbit or mice as compared to goat which is closely related to bovine for which BSA is self antigen. Different types of animals like rat , guinea pig , sheep , goat , horse elephant can be used to produce serum. The choice of the animal depends upon many factors such as amount of antigen available , degree of immune response, quantity of antibodies to be produced and maintenance of animals etc. 2. Property of antigen : This property depends upon size , shape, chemical composition of antigen. In general proteins of molecular weight higher than 5000 daltons and certain large polysaccharides are effective immunogens. Low molecular weight substance (hapten) could be linked to a carrier molecule before using it as immunogen. 3. Dosage of immunogen : Antigen or immunogen should be injected in optimum amount as high dosage results in tolerance of lymphocytes to antigen. Low dose results in poor immune response as sufficient number of lymphocytes are not activated. 4. Immunization route : Route of immunization also affects the immune response and thereby the quantity of antibodies produced. Antigen can be injected by various routes like (i) Subcutaneous (ii) Intra dermal (iii) Intraveinous (iv) Intra peritoneal (v) Intramuscular (vi) Foot pad route. Immunogen injected by these routes reach different secondary lymphoid organs and since the population of B and T lymphocytes and antigen presenting cells are different in these organs , it results in differential immune response 5. Adjuvants : Adjuvants are the substances which enhance the immune response by attracting the macrophages at the site of injection. This results in formation of granuloma. They also increase antigen persistence in system by enhancing the slow release of antigen from the site of injection. Adjuvants,commonly used are of two types : (i) Freunds complete adjuvant : This contains mineral oil , emulsifying agent (detergent (mannide mono-oleate) and killed Mycobacterium tuberculosis.

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(ii) Freunds incomplete adjuvant : This contains only mineral oil and emulsifying agent detergent (mannide mono-oleate). 6. Duration of immunization : First injection of immunogen is generally given in Freunds complete adjuvant.This results in primary immune response. After sometime , it is necessary to give a second booster dose of the same antigen in Freunds incomplete adjuvant. Booster dose results in secondary immune response which is more pronounced than the primary immune response. Therefore it is necessary to make a schedule of immunization before doing it.

MATERIAL REQUIREMENTS : Syringe (1ml/2ml), rabbit, cotton swab, alcohol, antigen solution, NaCl, Fruends complete and incomplete adjuvants
Solution : 0.85% NaCl

PROCEDURE A. Immunization schedule

1st Injection 2nd Injection 3rd Injection 4th Injection 5th Injection : : : : : 0 day 7th day : 14th day 21st day 28th day : : : : S.C. at multiple sites S.C./I.P. S.C./I.P. S.C./I.P. S.C./I.P.

B. Antigen preparation
1. Take 500 g of antigen in saline (1ml) 2. Add this drop wise to 1ml of fruends complete adjuvant. Homogenize it with the help of needle and syringe till it becomes like a white cream. 3. Finally a stable water in oil emulsion is obtained. This is checked by gently placing a drop in saline from syringe. If the emulsion is stable the drop will not disperse.

C. Immunization
First Injection : inject about 1ml of emulsion. In subsequent immunizations, the antigen is emulsified with fruends incomplete adjuvant and injected.

D. Collection of antiserum
1. 2. After 4 days of last dose of antigen, rabbit can be bled from the marginal vein of the ear. Lateral margin of the ear is shaved and cleaned with 70% alcohol. A table lamp may be placed beneath the ear to have a good look at vein. The vein may be dialated either by rubbing the ear or by applying cotton swab moistened with xylene.

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3. 4. 5.

With the help of a sharp blade a diagonal incision is made across the vein, and the blood is collected. After the collection of blood, bleeding can be stopped by pressing the vein at site of cut with dry cotton. Separate the sera from the blood and store it in aliquots at 200C. Check the titer of antisera by SRID

Objective : Principle: Requirements

EXERCISE NO.27 Extraction of IgG from serum. Gamma globulin is a very powerful antigen (a strong immunogen), and is usually prepared as an ammonium sulphate precipitate of whole serum. It is not strictly -globulin but is serum depleted mainly of albumin. Ammonium sulphate, dilute ammonia solution, blood, double distilled water (ddw), NaCl, centrifuge, glass rod, dialysis tubing, NaHCO3, EDTA, KCl, Na2HPO4, K2HPO4, BaCl2

Solutions 1. Saturated (NH4)2 SO4 solution: Dissolve 100 g (NH4)2 SO4 in 100 ml ddw at 500C, allow to stand at RT O/N and adjust the pH to 7.2 with dilute NH3. 2. Normal saline: Dissolve 0.85 g NaCl in 100 ml of ddw. PBS (phosphate buffer saline) pH 7.2, 0.15 M, 100 ml NaCl 0.8 g KCl 0.02 g Na2HPO4 0.115 g KH2PO4 0.02 g 4. 2% NaHCO3 + 0.05% EDTA solution Dissolve 2 g NaHCO3 and 0.05 g EDTA in 100ml ddw. 5. 1% BaCl2: Dissolve 1g BaCl2 in 100 ml ddw. Procedure A. Separation of serum from blood 1. Allow the blood to clot in the tubes, either in sunlight or in incubator at 370C. Free the clot from the walls of the tube to aid retraction. 2. Collect the serum by a pipette and spin it at 3000 rpm for 15 min.

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B. Precipitation of globulins from serum 1. Dilute serum 1:2 with saline and add saturated (NH4)2 SO4 solution to final concentration of 45% (v/v). Calculation for volume of saturated solution required to achieve a required concentration of (NH4)2 SO4 Volume of saturated solution (ml) to be added per 100 ml volume 100 (Sf-Si) = 1-Sf Where, Sf is Final saturation and Si is Initial saturation 2. Stir at 40 C for 30 min. 3. Centrifuge the ppt at 3000 rpm/30 min/40C. 4. Wash the precipitate with 45% saturated (NH4)2 SO4 solution and recentrifuge. 5. Redissolve the precipitate in the same volume of PBS as the original serum. Centrifuge to remove any insoluble material. 6. Re-precipitate the globulin using a final concentration of 40% saturated ((NH4)2 SO4) solution. 7. Spin off the precipitate and wash the pellet with 40% saturated (NH 4)2 SO4 solution. 8. Re-dissolve the pellet in minimum volume of PBS. 9. Dialyze the globulin isolated from serum, against 4 changes of PBS at 40 C. C. Activatioin of dialysis tubing 1. Select dialysis tubing of suitable diameter and length according to sample volume. 2. Submerge it in the solution of 2% NaHCO3 + 0.05% EDTA and boil for 10 min. 3. Discard the solution and again boil it in ddw for 10 min. 4. Activated dialysis tubing can be stored at 40C upto 3 months. D. Desalting of protein sample by dialysis 1. Seal the dialysis tubing from one end by a thread. 2. Pour the sample inside it using pipette. 3. Seal it from other end also. 4. Hang it in a beaker containing 100 volume of PBS. Keep the beaker on magnetic stirrer and leave it at 40 C at least for 2 h. Like this at least 4 changes are given. 5. To check whether dialysis (desalting) is complete or not add 1% BaCl 2 solution in the PBS. If precipitate appears then dialysis is incomplete and if no precipitate appears, dialysis is complete. E. Protein estimation 1. Determine the absorbance of the sample at 280 nm in a UV spectrophotometer. 2. At 280 nm, an OD of 1.0 (1 cm cuvette) is equivalent to an r-globulin concentration of 0.74 mg/ml.

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