Lysis buffer for membrane proteins and coimmunoprecipitation

Phoenix ddboyboy at she.com
Thu Nov 28 12:47:58 EST 2002


Mr John Ladasky,

I would like to ask if this lysis buffer can be applied for western blot?
And how many volume should i added to the cell and incubate for how long?
Also, can i be applied to homolgenized whole tissue?

Regards,
Phoenix



"John Ladasky" <ladasky at my-deja.com> ?????
news:c09b237b.0211081511.dcbac59 at posting.google.com...
> 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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