optimum conditions for transfer of ~20-30 kDa protein - (Dec/24/2008 )

I am having trouble in transfering 20-30 kDa protein on both nitrocellulose (for doing immunoblot) and on PVDF membrane ( for sequencing). I have a old wet bio-rad western blot appartus.

I am using 20% Methanol along with TRIS and Glycine in my transfer buffer, and wenever I do transfer, I see only 10% of the protein transferred on the membrane. Rest remains on the gel.

Transfer conditions are

45 Volts for 1h followed by 60 Volts for 1 h.

Since I have to use anti-His mAb, I need 100% of my expressed protein to be transferred for immuno detection.

Please advise.

Thanks,
jhil

I work with a 25KDa protein but I use semi-dry electroblotter. in case you can find a Biorad semi-dry electroblotter try using Towbin's buffer, use 3 filters down, 3 filters on top, use PVDF but activate it in methanol for 1-2 min prior to use (I hate Nitro), run on 15V and 60mA. it works fine for me.