Western blues

Michael Witty mw132 at mole.bio.cam.ac.uk
Sat Sep 23 12:11:24 EST 2000


Dear Deepika, my gels often show up protein after blotting (I don't know
about anyone else).  I have thought that proteins migrate differently when
blotting, so I ignore this and just pay attention to the membrane after
staining.

BUT if your PARTICULAR  protein does not transfer, how about this idea for
everyone to critisize: I think that the role of methanol in the transfer
buffer is to partially precipitate protein (so it will migrate slow and
not shoot through the membrane - nobody told me, it is my own idea and so
critize freely).

IF this is true, maybe your large protein is ending up too high molecular
weight to transfer easily.

SO, how about repeating the western using buffer without methanol?

Mike.


 On 23 Sep 2000, deepika wrote:

> 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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