Western Blot

Jim Hutchins hutchins at fiona.umsmed.edu
Tue Aug 29 07:27:50 EST 1995

Alison Weaver (a.weaver at auckland.ac.nz) wrote:
: I am having problems with the transfer of a 150KDa protein which 
: has a transmembrane domain from SDS PAGE to Hybond C membrane. 
: Does anyone have any suggestions how transfer may be enhanced? 

Along with the suggestions of others (lower MeOH, maybe some SDS) you
might also try two other things:

1. Blot in 10 mM CAPS buffer (Matsudaira, 1989)

2. Use a different membrane, e.g. PVDF.

Also, you did not mention your transfer voltage.  In a big blotting
chamber (BioRad TransBlot), we use 70 V for 1 hour in CAPS.  This seems
to work better than 14 V overnight at 4 deg C.

Note that CAPS is preferred (I did not mention, but should have, that
it is pH'd to 11.0) because at this pH, *everything* moves away from
the negative electrode.  We have the poor luck to always be studying
highly basic proteins which do not blot well in standard Towbin buffer,
pH 8.3.  The pKa of CAPS allows it to have a good bit of buffering capacity
at pH 11.0, where other buffers have none.

Hope this helps

Jim Hutchins
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/    ***    E-mail:   hutchins at umsmed.edu
``It became necessary to destroy the town in order to save it.''--American
infantry officer firing on Ben Tre, Vietnam, 2/8/68

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