Lysis buffer for membrane proteins and coimmunoprecipitation
Denise Vogel
a coimmunoprecipitation. I am looking for a lysis buffer which contains
reagents which do not denature the antibody. Could somebody help me?
Thank you!
Wolfgang Schechinger
you might leave out reducing agents like BME, DTT or DTE as well as ionic
detergents e.g. SDS.
Regards,
Wo
---
John Ladasky
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
Dr Engelbert Buxbaum
> 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?).
Nuclei need some Ca and Mg for stability. In EDTA they tend to break up
more easily, and you get gluey DNA all over your sample.
By the way, DMSO is a better solvent for PMSF than propanol (long term
stability).
Calbiochem has a usefull little booklet on the use of their preparations
of formalin-fixed S.aureus cells, which includes buffer receipies for
IP.
Duncan Clark
<c09b237b.0211...@posting.google.com> on Fri, 8 Nov 2002,
John Ladasky <lad...@my-deja.com> penned the following literary
masterpiece:
>*PMSF stock solution: 50 mM PMSF in 2-propanol. Stable for at least
>three months at room temperature.
Providing the propanol is very dry?
I remember 20 years or so ago cadging dry propanol from the protease lab
next door specifically for PMSF. Can't remember what they used to stick
in the Winchester to keep it dry - some sort of brown pelleted stuff -
I know, not much use without a name :(
Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
Duncan Clark
GeneSys Ltd.
Wolfgang Schechinger
>I know, not much use without a name :(
>
Molecular Sieves?
Wolfgang
>Duncan
>--
>I love deadlines. I especially like the whooshing noise they make as
>they go flying by.
>
>Duncan Clark
>GeneSys Ltd.
>
>
---
Phoenix
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" <lad...@my-deja.com> ?????
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