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CH 150: Introduction to Biochemistry SDS-polyacrylamide gel electrophoresis(SDS-PAGE)Introduction: Sodium dodecylsulfate (SDS) is a detergent that combines with and unfolds (denatures) proteins. If a protein is composed of subunits not covalently linked, SDS will disrupt the subunit interactions. SDS binds to proteins at a fixed ratio, about one molecule of SDS per two amino acid residues. This extensive binding of SDS to proteins dictates that the charge of the SDS-protein complex is constant regardless of the size of the protein, i.e., in an electric field all SDS-protein complexes will migrate toward the anode (+ pole) at equal rates if there are no other impediments to migration. In SDS-PAGE another impediment is introduced, a cross-linked polyacrylamide gel that has pores of limited size. This system allows separation of proteins based solely of the size of their subunits with smaller proteins or subunits migrating faster than larger ones. Comparing the migration of an unknown to that of proteins if known molecular weights allows one to estimate the molecular weight of the unknown. Normally, the migration, compared to the tracking dye, of standard proteins is plotted against the log of the molecular weights. This graph approximates a straight line and is used to estimate the molecular weight of the unknown based on the migration of the unknown in the same gel. SDS-PAGE differs from native-PAGE in two ways: (1) proteins treated with SDS are usually incapable of catalyzing an enzymatic reaction and thus activity staining in SDS-PAGE is not possible; and (2) proteins are separated solely based on mass with SDS-PAGE while with native-PAGE proteins are separated based on mass and charge density. Reagents: Pre-cast gels: Bio-Rad, 4-15 % acrylamide gradient gels Electrode
Buffer: 25 mM Tris 192 mM glycine 0.1 % SDS pH 8.3 with HCl Sample
Buffer: 62.5 mM Tris-HCl, pH 6.8 2% SDS 20 % glycerol 5 % b-mercaptoethanol Samples: 1mg/mL Alkaline Phosphatase 1 mg/mL Horse Radish Peroxidase 1 mg/mL b-Galactosidase 1 mg/mL human albumin Commercial
MW markers Sigma MW-SDS-7 dissolved in sample buffer:
Protein Stain: Pierce's Gel Code Blue Procedure: Sample Preparation:1. Dilute 20 mL of each sample in a labeled microfuge tube. Add 20 mL of sample buffer. Place these diluted samples in a boiling water bath for 5 minutes. Sample Application and Electrophoresis: 2. Before sample application, assemble the electrophoresis apparatus and gels. See attached appendix. Then add the electrode buffer. Add electrode buffer to the upper chamber such that the top of the gel is well covered. 3. Apply your samples (10 mL), one sample per well. Also apply a sample of the molecular weight markers (prepared as above). 4. Insert the electrode assembly into the electrophoresis chamber; fill the lower reservoir such that the bottom of the slab gel is covered. 5. Attach the lid to the apparatus and connect to the power supply and apply 25 mAmps of current (constant) per gel (i.e.- each set up should run at 50 mAmps constant current if two gels are on each electrode assembly). 6. Allow the electrophoresis to continue until the tracking dye (bromophenol blue) is about 1 cm from the bottom of the gel. At this point, turn off and unplug the power supply; disconnect the leads from the electrophoresis chamber to the power supply. 7. Disassemble the gels, placing the gel in about 100 mL of distilled water. Wash the gel 3 times for five minutes each time with distilled water. 8. Add approximately 100 mL of the staining solution (enough to cover the gel). Staining: 9. The gels will be stained by incubation with the protein stain for ~1 hr at room temperature. Once the gels are stained, they must be destained in distilled water to remove the dye from those areas not containing protein. Pour off the stain into the sink, being careful not to spill the gels. Add distilled water to the gel. The destaining process is also done at room temperature; addition of foam pieces or balled-up Kim-wipes speeds the process (10-15 minutes). Data
Sheets: Distance of dye front = _______________ cm Standards:
Samples:
Problems: 1. Calculate the relative mobility (Rf) of each band in the standards and your samples by measuring the distance each band traveled from the top of the separating gel, and then dividing this distance by the distance traveled by the dye front. Enter your answers in the data tables. 2. Using the molecular weight protein standards, plot log MW (y-axis) versus relative mobility (x-axis). Add a linear trendline; be sure to print the equation of the line and the correlation coefficient on the plot. 3. Using the standard curve, calculate the molecular size of each protein sample. Enter the answers in your data table. 4. What can you tell about the quaternary structure of each protein?
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