This article discusses the stabilization of multimeric proteins through disulfide linkages and the methodology for analyzing these complexes using non-reducing PAGE. The process ensures the structural integrity of proteins during electrophoresis.
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.
Prepare 1 liter of 1X Tris-glycine running buffer by mixing 25 millimolar Tris, 192 millimolar glycine, and 0.1% weight per volume SDS. Then, set up the SDS-PAGE running apparatus.
Open the 16% pre-cast TGX SDS-PAGE package per the manufacturer's protocol, and remove the cassette. Remove the comb that is lining the wells, and the tape from the bottom of the cassette, and place the gel into the running apparatus. Fill the chamber with the 1X running buffer, until the wells are submerged in liquid.
Using a plastic pipette, rinse out the wells with the running buffer. Load the samples onto the gel along with 10 microliters of the pre-stained standard marker. Finally, run the gel at 200 volts until the dye front is approximately 1 centimeter from the bottom of the gel.
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