native page - (Dec/22/2008 )

hi,
I am new in this line. I wish to know if anybody has done native page western blotting. I have tried many times. One more query is that how to study the mono. di or trimeric forms of a protein? Is their any simple technique? Thanks in advance.

what was your procedure?

what is the size(s) and pI(s) of your protein(s) of interest?

which native gel formulation (pH)?

what was the failure (didn't transfer, won't bind antibody, etc)?

Thanks
I have run the amples isolated from cells transfected with my construct in a PAGE without SDS (pH 8.8), then transferred it to PVDF membrane and probed with antiflag antibody. There was no separate band , only dark backgrounds are there. The size of protein is around 95 Kda.

did you stain the membrane with ponceau s to confirm transfer of protein?

did you stain the gel (after transfer) to see if any protein remained?

what is your transfer buffer? methanol is not necessary for transfer from native page unless you add sds (no more than 0.05%) to the buffer to aid in transfer of large proteins or to avoid excessive swelling of the gel during transfer (not really necessary for semi-dry transfer).

sorry if this is insulting, did you pre-soak the pvdf membrane in methanol to activate?

how do you block the membrane? the background should not be so high as you describe?

can you show us a picture?

if your protein is 95kDa and you want to look at dimers and trimers as well then you may want to add sds (and methanol) to your transfer buffer.

does your antibody recognize native or denatured protein or both? if only native then you must not use sds in the buffer.

just some thoughts.