detection of protein (20-30kd) by western

Nicole Rapior cbiei01 at uni-tuebingen.de
Fri Feb 13 21:22:41 EST 1998


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

Hello Didier,

with your small proteins it is possible that they just pass through your
nitrocellulose membrane. To prevent this use a double layer of
nitrocellulose and adapt your blotting conditions. We keep the current
constant depending on the size of the gel (5 mA/cm2). You also might
stop the blotting already after 20 minutes to prevent the proteins
passing through the membrane.

Good luck!!!!

Nicole
-- 
Nicole Rapior
Physiol. Chem. Institut
Hoppe-Seyler-Str. 4
D-72076 Tübingen

phone: 07071-297-3353
fax:   07071-29-3361
email: cbiei01 at uni-tuebingen.de



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