SDS-Polyacrylamide Electrophoresis
A. SDS-Polyacrylamide Electrophoresis Background.
Proteins can be separated on the basis of size alone if they are first solubilized with the
anionic detergent sodium dodecylsulfate (SDS). The SDS binds to the individual polypeptide
chains comprising the protein, converts the chains to rod-like shapes, and overwhelms the native
protein charge with uniform SDS negatively charged groups. When subjected to polyacrylamide
gel electrophoresis (PAGE) the polypeptide chains, which now have equal charge to mass ratios,
are separated primarily according to molecular size by the sieving effects of the gel. In SDSPAGE, migration distance correlates semi-empirically with molecular weight, and a straight line
should be obtained when plotting log [MW] vs. migration distance.
For further information on SDS-PAGE, you may consult the original reference: Weber, K.
and Osborn, M. (1969), "The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis", J. Biol. Chem. 244: 4406-4412 (1969).
B. SDS-PAGE Protocol.
Solutions Used:
Running Buffer: Purchase Pre-Made: Dilute to 1x
Protein Sample Buffer: Purchase Pre-made 2x
Molecular weight standards: known molecular weights of standards used will be provided by
your TA
Staining Solution: 0.5% coomassie brilliant blue R-250 in 50% isopropanol, 50% acetic acid
Destaining solution: 10% methanol, 10% acetic acid
Procedure:
Prepare samples for loading onto the gel
1. Dissolve 50 g/mL of protein in water (or buffer)
2. Add 50L of protein solution to 50L of sample buffer
3. Heat at 90ºC for 5 minutes
Assemble the gel running unit
1. Use pre-cast gel. Remove the comb gently, remove paper tab from bottom of pre-made
gel.
2. Insert gel into running apparatus. Your TA will demonstrate how to do this.
3. Add running buffer to the upper and lower portions of the assembly.
Loading and running the gel
1. Load 30 l of each sample into the well.
2. Load 10 l of known molecular weight standards in lane 1.
3. Connect the power supply to the gel running assembly with correct polarity, and run your
gel at a constant 150V.
5. Your gel will run for ~60 minutes.
6. During this time your TA will give a lecture on SDS PAGE.
Removing the gel and staining/destaining
1. Once the run is complete, disconnect the power supply. You TA will demonstrate how to
take the gel sandwich apart and transfer the gel into the staining container.
2. Add Coomassie Blue R-250 solution and stain for at least 20 minutes.
3. Remove the gel from the staining solution and place in destaining solution
4. Determine mw of proteins