Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate
Transcrição
Multimeric protein complexes are essential for various biological processes.
To separate protein complexes in their native state by multimer-PAGE, take tissue lysate containing the desired complexes. Load the lysate onto a gel assembly for Blue native PAGE.
Fill the system with an alkaline running buffer containing blue anionic dye and connect it to the power supply. At an alkaline pH, dye molecules bind to the protein complex imparting it an overall negative charge without denaturing. This negatively charged, intact protein complex migrates through the gel as a blue band.
Following a minimal run, and allowing the intact protein to migrate into the resolving gel, excise the strip containing the complex. Incubate the strip in a solution containing a cross-linking agent to allow its reactive groups to interact with proximal protein's amine groups, stabilizing the multimeric complex.
Replace the cross-linking solution with a quenching reagent to subside the unreacted cross-linkers' activity. Add SDS – an anionic detergent – which imparts a uniform negative charge to the intact protein complex while preserving their native size and makeup.
Place the treated strip in a fresh cassette and re-cast it into a new SDS-PAGE gel. During electrophoresis, the negatively charged cross-linked protein complex migrates toward the positive anode and appears as a high molecular weight band in the gel.
