Lysis buffer for membrane proteins and coimmunoprecipitation
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?
"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
> 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
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