How to optimise my western semi-dry transfer? - (Dec/04/2014 )

Hey,

I have ran my samples on one gel for coomassie staining and on another one for Western Blotting. Ponceau stained the membrane and post-transfer stained the gel. It seems like only half of the protein is transferred. Especially the marker gets transferred onto the filer, but the protein is lagging behind. Can anybody recommend what I should change?

Attached the images and details about the protocols.

Transfer: 1.5h @ 20V (current was hovering around 50mA most of the time).

Transfer buffer -

3.025g Tris Base

14.4g Glycine

100ml methanol (Sigma Aldrich, UK)

Add dH2O to final volume of 1L. pH 8.1-8.5.

gel: 4-12% Novex gradient, precast gel.

-Podge-

Hallo

I think your transfer process is quite ok.

-monekobe-

you can add up to 0.05% sds (and double the methanol to 20%) to the transfer buffer to improve efficiency (it's especially useful when transferring high molecular weight proteins).

-mdfenko-

thanks for the reassurance the transfer was ok.

Added the SDS and it looks better now, too.

Thanks

-Podge-