byung-hoon.kim at uni-tuebingen.de
Mon Sep 25 03:34:07 EST 2000
in my opinion you have used gels with too high % of polyacrylamid. For your
protein (160 kDa) I would recommend 5 - 6% PAGE gel. And in regard to the
transfer efficiency, I've never experienced the complete (100%) transfer.
> 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 ?
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