Isolation of cell membrane protein
Wolfgang.Schechinger at med.uni-tuebingen.de
Fri Nov 5 07:49:25 EST 1999
here comes your starter package for biochemistry:
Use 0.5 to 1% (vol) TX100 or NP40 (probably any nonionic detergent
works) in a suitable buffer (PBS without Ca2+ and Mg2+ or Tris or
what you like) together with suitable protease and other
enzyme inhibitors (e.g. a cocktail e.g. from sigma, at least EGTA and
EDTA against metal ions, aprotinin, leupeptin, PMSF, mercaptoethanol
Incubate your cell layer 10 min at 4 deg C with this lysis buffer
(100 to 200 ul for a 3 cm dish), collect the supernatant, clear it by
centrifugation (10 to 30 min at full speed in a cooled tybletop
centrifuge (about 15.000xg))
If you have non adherent cells, pellet and wash them as usual, the
resuspend in lysis buffer, pipet five time upo and down. this should
be enough for most kind of cells.
If you have biologic material, homogenize it in lysis buffer.
The protocol probably doesn't work with plant cells due to different
The receipe dissolves membrane proteins but leaves nuclei and actin
in the pellet. If you don't want to see any cytosolic proteins, you
will have to use preparative ultracentrifugation first, using a
density gradient (e.g. with percoll from pharmacia). You might get
protocols from pharmacia or from your library. There's a book on
ultracentrifugation from oxford university press.
Enjoy and have a nice weekend!
> From: kyasuda at pharm.kyoto-u.ac.jp (Kei Yasuda)
> Subject: Isolation of cell membrane protein
> Date: 3 Nov 1999 23:29:12 -0800
> Organization: BIOSCI International Newsgroups for Molecular Biology
> Reply-to: kyasuda at pharm.kyoto-u.ac.jp
> To: methods at hgmp.mrc.ac.uk
> Please teach me the protocol of isolation of cell membrane proteins.
> I don't know about biochemistry well. After isolation, I want to
> analyse on SDS-PAGE.
usual disclaimers apply
Dr. Wolfgang Schechinger
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de
*unsolicited mail is *NOT* appreciated
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