Why not? You could try to soak the native gel in a solution with strong denaturing agent, wash and then do the transfer. However, your protein will not be "linear" and your antibodies might still fail to recognize anything. Perhaps adding SDS to the transfer and denaturing solution will help.

Why not? You could try to soak the native gel in a solution with strong denaturing agent, wash and then do the transfer. However, your protein will not be "linear" and your antibodies might still fail to recognize anything. Perhaps adding SDS to the transfer and denaturing solution will help.

 for western blotting,  protein after SDS-PAGE must bind to transfer membrane used (e.g.nitrocellulose membrane). I agree with above answer. but binding affinity of native and denatured protein may be differ.

Hi Philippe,

I agree with the answers above but have you also considered that your antigen (i.e. the AB recognition site) on your protein is exposed? It might be that due to the correct folding/multimerisation of your protein your AB can't bind.

Cheers,

Karim

Why don't you just cross-link your samples and run a regular SDS-PAGE? You should still see oligomerization and your proteins will be denatured for your Ab recognition.

We use transfer buffer with 0.02% SDS however that still does not allow full denaturation, and many of our antibodies fail to work on a BN PAGE blots. What we often do is a second dimension SDS PAGE. This is laborious, but if other methods fail, you could try it out. Basically you cut a lane of the BN PAGE and lay it horizontally at the top of the SDS glass plates. Then pour SDS gel and run it as usual.

Dear Philippe, Did you ever solve the problem? I too have the same issue, I have a protein that oligomerizes that I can see on Native page but then I want to use an antibody some how on this native gel.

Thank you