How to analyze antigen-antobody complex on gel? - Which gel will NOT break the complex? (Nov/25/2005 )
Hi, just need a word of advice
I'd like to analyze a complex consisting of protein of interest and antibody against it by electrophoresis. I know that conventional SDS-PAGE will disassemble such a complex.
Has anyone done something like this?
Would dropping the boiling step and eliminating SDS from gel help it?
Would it be possible to stabilize the complex by some kind of cross-linker between the antigen and antibody?
Now there are some details:
Protein of interest: oligomer of yeast prion Sup35NM
Antibody: generic antioligomer antibody A11 (Glabe' 04)
Well, as you want to see the interaction you need a non-denaturing of native gel.
So it seems to me you should NOT use SDS and heating. If you really need a detergent perhaps non-ionic would be better (such as Triton X-100 or Tween-20...).
I suggest you look at protocols for protein gels in non-denaturing conditions (or perhaps for gel shift assays too). I would bet there are well-tested methods for the type of experiments you would like to do.
Like Serge was saying same recommandations and also no Beta mercapto ethanol or DTT in the sample buffer cause it disrupt the association of certains protein complexes
A pre-cast Tris-HCL gel would probably work. Bio Rad do a good one, it dosn't contain any SDS and the HCL is there to correct the pH so shouldn't denature your protein.