Western blues

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Mon Sep 25 12:43:08 EST 2000


This message has been posted by:  612 right <i.hinners at REMOVE-THIS-TO-SENDicrf.icnet.uk>

I also think you should use a lower % acrylamide. I am working with a 100
kDa protein that already transfers like shit in a 10% gel but transfers
much much better in a 7.5 % gel....
Adding SDS should also help, but be careful, just because the stained gel
contains less protein after blotting, in my experience doesnt mean there's
more on your membrane, so, also check the membrane.

Hope that was of any help, regards, Ina


deepika wrote:

> Mike, I think, traditionally methanol was part of the transfer buffer
> because
> methanol helps proteins bind to nitrocellulose. I am not sure that
> the same logic applies to PVDF.... but it is in the buffer nonetheless.
>
> I have noticed that when I lower the MeoH concetration the current
> flowing through reduces i.e. for the same voltage (100 V) I get 200
> mAmp instead of 250 mA. So my question is does voltage matter more than
> current as far as transfer effficiency of high mol wt protiens ? or vice
> a
> versa ?
>
> Lee, I have not added SDS to the transfer buffer yet...I read somewhere
> that 0.05 % was enough....
> Deepika
>
> ---

--
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hinners at icrf.icnet.uk







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