Protein electrophoresis: Introduction to SDS-PAGE

Transcription

1 Protein electrophoresis: Introduction to SDS-PAGE

2 Aim: -Separation of proteins in an electric field by electrophoresis. Purposes: -Estimation of molecular masses -Relative abundances of major proteins in a sample

3 SDS-PAGE = Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis. SDS-PAGE uses a polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. Also called the Laemmli method, for first application. Laemmli UK, Nature 1970, 227 (5259): Cleavage of structural proteins during the assembly of the head of bacteriophage T4..

4 Laemmli UK, Nature 1970, 227 (5259): Cleavage of structural proteins during the assembly of the head of bacteriophage T4..

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6 SDS is an anionic detergent. A polypeptide chain binds SDS in molar proportion to its mass. SDS denatures most complex structures of proteins. It is also attracted toward the positively-charged anode. About 1 SDS binds per 2 AA.

7 Polyacrylamide gels restrain larger molecules from migrating as fast as smaller ones. The separation of proteins depends almost entirely on the differences between the molecular masses of polypeptides. In a uniform gel, the migration distance of a protein (R f ) is proportional to the -log of its mass. Proteins of known masses are run along with the unknowns. This allows to plot R f vs. mass, and to estimate the masses of unknown proteins.

8 Stacking gel - Heavier protein Separating (or Resolving) gel Lighter protein +

9 Polyacrylamide gels for SDS-PAGE Important parameters: -percent total acrylamide in a gel -relative percentage and type of crosslinker -gel dimensions Manual preparation requires casting two different layers of acrylamide between glass plates. Lower layer: separating gel. Upper layer: stacking gel with sample wells. Stacks proteins into micrometer thin layers.

10 SDS Gels -A 7% acrylamide gel separates proteins with masses between 45 and 200 kda (no resolution below 45 kda). -A denser gel, e.g. 14%, resolves smaller polypeptides. It would not separate proteins above 60 kda. -To analyze the entire profile of a sample containing heavy and light polypeptides: run two gels.

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12 Running Gel: Buffer stock solution: 0.4% SDS, 0.5 M Tris-HCl, ph 6.8. Buffer, linker and acrylamide stock solutions are mixed to obtain from 7 to 15% of acrylamide monomer (+ variable conc. of crosslinker). Acrylamide polymerizes in the absence of O 2 : the mixture is placed under vacuum for 5 min. Then polymerization occurs quickly. The solution is poured into a cassette.

13 The gel mix is poured, then overlaid with water-saturated butanol. This will produce a smooth, leveled surface to keep bands straight and uniform.

14 Stacking gel Stackers have 3-4.5% acrylamide (low density) to enable very large proteins to run. At polymerization, the mix is poured into the cassettes on top of the separating gel. A comb is inserted and adjusted evenly.

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16 Protein + sample denaturation

17 Protein denaturation prior to SDS-PAGE Sample is mixed with 2% SDS, 20% glycerol, 20 mm Tris-Cl, ph 8, 2 mm EDTA, dithiothreitol (DTT), and bromophenol blue as a tracking dye.

18 Roles of denaturing components EDTA chelates divalent cations, then reduces the activity of enzymes that require Ca 2+ and Mg 2+ ions. Glycerol makes the sample denser than the buffer: sample will remain at the bottom of a well rather than float out. Dye: tracks the progress of electrophoresis. SDS: adds negative charge to the amino acids. Proteins are straightened, thus functionless. DTT breaks covalent disulfide bonds.

19 Cystine contains a sulfhydryl (-SH) group that spontaneously forms a disulfide bond (-S-S-) with another sulfhydryl group under normal intracellular conditions. DTT is a strong reducing agent for disulfide bonds.

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21 SDS-PAGE yields reproducible results, but provides low precision for MW determination. Amounts to load A typical mini-gel well holds 10 µl of a 2 mg/ml solution of denatured protein (0.02 mg or 20 µg).

22 Running gels The anode (+) must be connected to the bottom chamber and the cathode (-) to the top chamber. Gels are run at a voltage (typically 150 V) that will bring the tracking dye to the bottom as quickly as possible without overheating the gels.

23 Disassembly and staining When the dye front is near the bottom of the gel, the run is stopped. Plates are separated and the gel is dropped into a staining dish. Staining usually requires incubation overnight, with agitation.

24 Staining protein gels Commonly used: 0.1% Coomassie Blue dye in 50% methanol, 10% glacial acetic acid. Acidified methanol precipitates the proteins. The dye inflitrates the entire gel, however only sticks permanently to proteins. Excess dye is washed out by 'destaining' with acetic acid/methanol, also with agitation. Gels can be dried down, cut or photographed for later analysis and documentation.

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26 Steps to analyze the gel Calibrate the gel using standards of known molecular mass Estimate molecular mass for each band of interest Note differences in intensity of staining that reflect abundance of individual proteins Note unusual patterns that might indicate incomplete denaturation, degradation, etc.

27 Estimate apparent molecular mass for unknowns The relationship between mass and R f is logarithmic. Data are interpolated from the standard curve. High molecular mass: -myosin (205,000) - from rabbit muscle -beta-galactosidase (116,000) - from Escherichia coli -phosphorylase B (97,400) - from rabbit muscle -bovine serum albumin (66,000) -egg albumin (45,000) -carbonic anhydrase (29,000) - from bovine erythrocytes

28 Low molecular mass -bovine serum albumin 66,000 -egg albumin 45,000 -glyceraldehyde-3-phosphate dehydrogenase (36,000) - from rabbit muscle -carbonic anhydrase (29,000) -trypsinogen (24,000) - from bovine pancreas -soybean trypsin inhibitor (20,100) -alpha-lactalbumin (14,200) - from bovine milk

29 205k 66k 116k 97.4k 45k 36k 29k 20.1k 24k

30 Measuring relative mobility of protein bands Relative mobility is the distance migrated by a band divided by the distance migrated by the dye front. It is used to compare the migration of a protein from gel to gel, regardless of the physical length of the gel or duration of electrophoresis.

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33 How gel concentration affects relative mobility Acrylamide concentration determines the cut-off at the low end. All polypeptides below a minimum mass run with the tracking dye. Resolution of individual bands diminishes near the top of a gel. Usually the top 10% or more of a gel is unusable. A gel with low density resolves larger polypeptides while cutting off lighter ones. A gel of higher density will reveal smaller polypeptides, while compressing and possibly distorting the larger ones.

34 3 = phosphorylase B (92,000)