Transmembrane proteins aggregate in SDS-PAGE?
ronni at ucla.edu
Tue Aug 7 21:45:37 EST 2001
I would be grateful for any advice concerning the following.
I am overexpressing FLAG-tagged transmembrane receptors, e.g.
mCD4 and mCD4/TLR4 fusion with pFLAG-CMV-1 vector. Transfected
293T cells show robust signal compared to mock transfected cells in
FACS with anti-CD4 PE staining. When I make Western samples from cells
from *same transfected plates* I see strong (and bit smeary) FLAG
signal but it is above 150 or even above 200 kD. I use 150 mM NaCl, 50
mM Tris-HCl pH 7.5, 1% NP-40 and 1 mM EDTA to lyse the cells (30 min
on ice, spin, take sup).
Expected signal is around 50 kD with mCD4 or 65 kD with mCD4/TLR4.
It looks like the proteins are aggregating? I tried fresh
2-mercaptoethanol and incubating samples in RT, 37C, 70C or 100C. Also
tried different amounts of total protein. No effect. Still see the
slow-migrating bands. Any ideas?
Tapani Ronni, Ph.D.
UCLA School of Medicine
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