Immunoprecipitation in Non-reducing conditions? - Protocol suggestions? (Oct/04/2006 )
Is there was a way to do an immunoprecipitation under non-reducing conditions? My protocols have always involved boiling the beads in SDS-PAGE sample buffer with DTT, but was hoping we could liberate our antigen in some way without using the reducing agent. I would greatly appreciate any information you could supply!
you may be able to release the antigen by changing the pH to 2.5 or 9.5 to 10 as you would for immunoaffinity chromatography.
Boiling the sample without DTT will separate the anitgen from the antibody, and the antibody from the beads.
The binding is non-covalent so SDS alone should destroy any bond formation.