Non-Reducing SDS PAGE: A Method to Analyze Disulfide-Linked Multimeric Protein Complexes

Multimeric proteins are stabilized by inter-molecular, covalent, disulfide linkages between their cysteine residues which is crucial in maintaining their structural and functional integrity.

To analyze such complexes by non-reducing PAGE, take the desired concentration of disulfide-stabilized proteins. Add a non-reducing sample buffer and heat the sample.

This buffer contains SDS, which imparts an overall negative charge to the proteins, and a tracking dye to visualize their movement in the gel. The buffer lacks any reducing agent, which allows the disulfide bonds to remain unimpaired, facilitating the multimeric complex to remain intact.

Load the individual complexes into wells of the polyacrylamide gel of the desired percentage, pre-assembled in a gel apparatus. Add a protein ladder of known size in another well. Submerge the wells in an SDS-based running buffer and connect the apparatus to a power supply.

The electric current forces the negatively charged protein complexes to move through the pores of the gel toward the anode – the positive terminal. Since the protein complexes are uniformly negatively charged, the separation occurs based on size, where the multimers form a prominent higher molecular weight band at a specific position.

Compare the size of the separated protein band with the ladder. The absence of lower molecular weight bands in the gel confirm the successful separation of the intact multimeric complexes.