detection of protein (20-30kd) by western

Sam Lee samuel.lee at utoronto.ca
Fri Feb 13 10:59:11 EST 1998


Hi Didier,

Perhaps your transfer is the problem. After your transfer, stain your
gel (using Coomassie Blue, for example) and determine if there is a lot
of protein remaining in the gel. Also, if you use a prestained molecular
weight ladder, you can see if your transfer is efficient. 10 V for 30
minutes seems a little low to me. We transfer minigels on a semi-dry
transfer system (though it's not BioRad's) and our conditions are 30 V
for 1-2 hours.

Sam.

------------------------------------------
samuel.lee at utoronto.ca

Department of Pharmacology
University of Toronto
Toronto, Ontario
Canada



Didier Fesquet wrote:
> 
> hi, I am having trouble to detect protein in the range of 20-30 kd by
> western blotting. I am tranferring the gel onto Nitrocellulose(0.22um)
> as well as immobilon P using semi dry tranfer (biorad apparratus) and
> the recommended voltage 10volt , 30min for a mini gel. upon transfer, by
> ponceau staining , I could not detect sharply stained protein band. what
> happen!
> any help is wellcome
> 
> thanks
> 
> didier



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