Isolation of cell membrane protein

Wolfgang Schechinger Wolfgang.Schechinger at med.uni-tuebingen.de
Fri Nov 5 07:49:25 EST 1999


Dear Kei,

here comes your starter package for biochemistry:

Lysis buffer
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 
or DTT).

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 
cell walls.

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!

Wo


> 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   
Pathobiochemistry Dept.      
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de 
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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*unsolicited mail is *NOT* appreciated
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