SDS-PAGE

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis or SDS-PAGE is a molecular weight-based technique that separates mixtures of proteins based on their size.

Here, sample proteins are treated with β-mercaptoethanol and SDS.

β-mercaptoethanol is a reducing agent that breaks the disulfide bonds.

SDS, a negatively charged detergent, binds at the hydrophobic regions to unfold and denature the proteins and adds a uniform negative charge.

The samples are then loaded onto a polyacrylamide gel made of crosslinked acrylamide monomers.

The top stacking gel has large pores due to low acrylamide concentration.

On voltage application, the protein samples stack like coins to enter the lower resolving gel simultaneously.

The resolving gel has smaller pores due to a higher acrylamide concentration. It acts as a molecular sieve, allowing smaller proteins to run faster, thus separating the proteins based on their sizes.

These separated protein bands are commonly visualized using coomassie blue staining. The molecular weight of each band can be determined using reference marker proteins.