Lysis buffer for membrane proteins and coimmunoprecipitation

John Ladasky ladasky at my-deja.com
Fri Nov 8 18:11:25 EST 2002


Denise Vogel <denise.vogel at molbio.unizh.ch> wrote in message news:<3DCBCBAC.FE962CB9 at molbio.unizh.ch>...

> I have to isolate a membrane protein from transfected HEK293 cells to do
> a coimmunoprecipitation. I am looking for a lysis buffer which contains
> reagents which do not denature the antibody. Could somebody help me?
> Thank you!

Greetings, Ms. Vogel,

I prepare immunoprecipitates of the transmembrane protein Bap31 in
HeLa cells, so my situation is quite similar to yours.  I use the
following lysis buffer.  It does no apparent harm to antibody
molecules.

   NaCl                         0.15M
   Tris-HCl, pH 7.5             0.050M
   Triton X-100                 0.50% (v/v)
   PMSF solution*               2.0% (v/v)
   other protease inhibitors**  8.0% (v/v)

*PMSF stock solution: 50 mM PMSF in 2-propanol.  Stable for at least
three months at room temperature.

**Stock solution of other protease inhibitors: we use Roche "Complete"
EDTA-free protease inhibitor cocktail tablets (of course, I have no
affiliation with Roche).  Dissolve one mini-sized tablet into 2.6 ml
sterile water.  Stable for one week at 4C.

Even though the Ab molecules are fine in this buffer, we have learned
that co-IP of Bap31 and one of its partners, namely class I MHC
molecules, is disrupted.  This may be relevant to your results.  One
of my colleagues has obtained co-IP between Bap31 and class I MHC by
switching to CHAPS or digitonin-based lysis buffers.  If this
information would be useful to you, please let me know and I will ask
my colleague to provide me with details.

In the lab where I worked previously, the lysis buffer used for
protein chemistry included EDTA, which for some reason isn't used
here.  I don't believe that the addition of EDTA would affect the
results with most proteins.  EDTA also offers the advantage of being
able to prepare a stock solution which does not become contaminated. 
In fact, in this quirky procedure that I'm using, EDTA is added later
in the process, after the nuclei have been spun out of the lysates.  I
am not sure of the reason for this (would anyone care to comment?).

Good luck!

--
John J. Ladasky Jr., Ph.D.
Department of Biology
Johns Hopkins University
Baltimore MD 21218
USA
Earth



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