Protein A-antibody SDS-PAGE

Question

I wanted to know whether the boiling of Protein A- antibody complex for SDS-PAGE analysis will disrupt the interaction? and result in dissociation of protein A and antibody?

Thank you

Answer 1

Yes. Samples for SDS-PAGE are mixed with a loading buffer containing SDS (final concentration of SDS is usually 2%). Generally, the SDS will disrupt protein-protein interactions, even before boiling. Reducing agent (usually DTT or b-mercaptoethanol) is added to disrupt disulfide bonds. See diagram here.

There are, however, exceptions. Some protein complexes/multimers are resistant to SDS until boiling, and some even maintain interactions after boiling. Some examples: Xia et al 2007, Kubista et al 2004, Grigorian et al 2009.