Western Blotting of Membrane Proteins in the presence of SDS

James B. Hutchins jbh at anat.UMSMED.EDU
Fri Apr 23 20:20:13 EST 1993


In article <01GXBS80C7B6003K5N at nic.the.net> SHAUN%JASON.DECNET at relay.the.net
("Shaun D. Black") writes:
...stuff deleted...
>what [conditions] others have found optimal for 
>membrane proteins from SDS-PAGE -> PVDF.
>
>     1. Buffers:  [50mM Tris / 380mM Glycine] vs [25mM Tris / 190mM Gly]
>                  (or others?)

Depends on the protein.  We have had success with two buffers: original
Towbin buffer (25 mM Tris, 192 mM glycine, I think, 0-20% MeOH,
pH 8.3) and Matsuidaira CAPS buffer (10 mM CAPS, 10% MeOH, pH 11.0).
See below. 

>     2. SDS    :  0% vs 0.1% (or others?)

None for us.  It doesn't seem to help, and definitely hurts in many cases.

>
>     3. Methanol: 20% required?

Depends on the mol.wt. of the protein in question.  Below 100 kD or so,
10-20% MeOH is good; above, we grade down the MeOH.  1% is good for proteins
about 200 kD.

>
>     4. Voltage / Time:  Is there an optimum or do membrane proteins simply
>                         require more time?
>

For Towbin buffer, we use 14 V overnight 4 deg C.  For Matsuidara/CAPS
buffer, it seems to degrade overnight so we use 70 V 1 hr room temp.

>     5. Is it important to blot right after SDS-PAGE or can gels be soaked
>        in blotting buffer overnight before electrotransfer?

I would definitely NOT recommend an overnight soak.  Proteins will tend
to diffuse within the gel (or out of it!) and form smeary (or absent!) bands.

Two very good references, incomplete because I am writing at home, are
van Oss and Chaudury (1987 I think) and Otter T Anal Biochem 1989 or 90.
The first explains the theory behind Western blotting and the second
describes a protocol for efficient transfer of proteins over a wide MW
range.  Plus Tim Otter is a nice guy.

Basically, all Western blotting depends on partial renaturation of the
proteins on the membrane to a state where the antibody (generally
developed against the native protein) can recognize its epitope.  The way I
see it, the reason monoclonals so often fail to work in Westerns is that one
may not renature the protein to recreate the singular, original epitope to
which the antibody is directed.  As demonstrated in many systems, there is
no "law" which says that a protein must reform its original, native
configuration after denaturation: the low-energy form was created by a
speeding ribosome in the endoplasmic reticulum, not in Towbin buffer.
Just ask a fried egg.

If there is sufficient interest, I will look up the references and
post them to the net.  Just e-mail me.

>  = Shaun D. Black, PhD     | Internet:  shaun%jason.decnet at relay.the.net = 

Hope this helps.
Jim


-- 
Jim Hutchins                    []     E-Mail: jbh at anat.umsmed.edu
Dept of Anatomy                 []
Univ Mississippi Med Ctr        []



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