Western blues

David J. Meyer, Ph.D. meyerdj at phibred.com
Mon Sep 25 15:36:11 EST 2000


I agree that a lower %T is indicated. Others have suggested the adding SDS
to the transfer buffer may help--also true.

You may consider instead using the transfer buffer of Matsuadaira
(Matsudaira, P. 1987. Sequence from picomole quantities of proteins
electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem.
262:10035-10038) which is 10 mM Na-CAPS, pH 11.0, 10% MeOH. Because this
buffer is rather more basic than the usual Towbin buffer, your proteins
likely will have a greater net negative charge. I have observed better
transfer of many different proteins using this buffer. Be careful to use
cold buffer, if possible, as this one has enough salt in it to generate a
lot of heat during transfer!

Hope this helps.

David J. Meyer, Ph.D.
Pioneer Hi-Bred International, Inc.
deepika wrote in message <39CF6538.97FF5694 at umich.edu>...
>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
>
>
>---







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