Western blues

deepika deepika at umich.edu
Sat Sep 23 10:06:23 EST 2000


Hi
I have been having a lot of problems with efficiency of transfer from
gel (10% acrylamide/bis) to PVDF membrane (Immobilon P). When I stain
the gel after transfer, there is a lot of protein left on there. I know
that I am not exceeding the binding capacity of the membrane (I did a
titration of ug protein loaded and discovered that the ratio of protein
transferred to those that stay behind is the same).
            The protein of interest is high moleculat weight (around 160
kDa). I have tried :
- reducing MeOH concetration in the buffer from 20% to 10 %.
- doing an O/N transfer (25 V) followed by 1/2 hr 100 V
- doing a 2 hr 100 V transfer

Nothig really helps. Somethimes there is enough protein for me to see (I
use LumiLight, Roche) and sometimes there's not. So help, please !! Are
high mol wt proteins transferred better at high or low field strength ?
Has anyone tried SDS in the transfer buffer ?

Thanks
Deepika


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