Removal of lipids from cell lysate?

Kevin Mulcahy k.mulcahy at sheffield.ac.uk
Tue Feb 10 06:08:21 EST 1998


Hi all,

I am trying to purify a cell surface transmembrane protein by 
immunoadsorption of the cell lysate down a immunoaffinity column. The 
cells (1 x exp8) are lysed in 5 ml of 50mM Tris-HCl (pH 8.2), 150mM 
NaCl, 1% Nonidet P-40 for 40 mins on ice with occasional mixing. 
However, when I spin the cell lysate for 30 mins at 13,000 rpm (10,000 - 
12,000g) in a microcentrifuge at 4šC a milky white layer is present at 
the top of the supernatant which quickly disperses into the rest of the 
supernatant when I move the tube. I suspect this is lipids from the cell 
membranes. If so, then I will have to remove them before running my 
lysate down the immunoaffinity column otherwise they may physically 
block up the column and/or bind to the immunoaffinity sites in the 
column (thereby masking these sites from the protein of interest). Has 
anyone encountered this problem before? If so, could you please let me 
know how you removed the lipids before running the lysate down the 
column?

Many thanks for your help,

Kevin.

--------------------------------------
Dr Kevin A. Mulcahy
The Institute for Cancer Studies,
University of Sheffield Medical School,
Beech Hill Road,
Sheffield S10 2RX
Telephone:     +44 (0)114  271 2237
FAX:               +44 (0)114  271 3515  

Email Address: k.mulcahy at sheffield.ac.uk



More information about the Methods mailing list