problem transfering small proteins - (Jan/24/2006 )

Hi, friends!!!
I have a problem in transfering my protein, because it has a small size and I think I´m loosing her within the transfer process.
I´m using a semi dry trasnfer machine from bio-rad, and I wonder if somebody can advice me over the good conditions to transfer this kind of proteins...........
Thank you very much!!!!!!!!

how small?

what's the pI?

if it is similar to a POI I have worked with before, I may be able to help you...

what is your protocol?

I've not done semidry transfer, but a good rule of thumb is when blotting a small protein, use two sheets of nitrocellulose. In case the protein blows thru the first sheet it will get caught by the second one.

use nitrocellulose 0.2 um
boil membrane for 10 minutes after transfer

I would say, 0.2um PVDF, and fix it after transfer

but the note about two membranes is a big tip