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Electro-blotting of proteins

Andreas Weber aweber at biolan.uni-koeln.de
Wed Jul 27 07:49:32 EST 1994


In article <fodde.10.000CB663 at ruly46>, fodde at ruly46 (Riccardo Fodde) says:
>
>
>Hi everyone,
>
>I would like to ask whether anyone out there has a good protocol for the 
>electro-blot transfer of proteins. I would specifically like to know the 
>composition of the blotting buffer, type of membrane and running 
parameters. 
>The proteins that I would like to blot are in vitro synthesized 
biotinylated 
>peptides of up to 100 kDa. 
>
>Rob van der Luijt
>MGC-Dept. of Human Genetics
>Leiden University
>Leiden, The Netherlands
>Tel.:  31-71-276097
>Fax.: 31-71-276075
>E-mail: rob at ruly46.leidenuniv.nl

Hi Rob,

I would recommend 50mM Sodium-Borate/20%MeOH (pH 9,0) as anodic
buffer and 50mM Sodium-Borate/5%MeOH (pH 9,0) as cathodic buffer.
As membrane I would suggest Transblot from Biorad. If transfer is
not efficient, you can add 0,05% SDS to the cathodic buffer. Other
membranes then Transblot are ok, as long as you dont use SDS.
Blotting time is 3-4h at 1mA per cm2 of gel area.
Do not equlibrate the gel in blotting buffer.
References: Jungblut et al., Electrophoresis 11:581-588 (1990)
and Eckerskorn et al., Electrophoresis 1993/94, I dont recall.

Hope this helps

Andreas


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