Transmembrane proteins aggregate in SDS-PAGE?
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Aug 8 01:13:11 EST 2001
ronni at ucla.edu (Tapani Ronni) wrote:
>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?
Not an infrequent observation for glycosylated TM proteins.
I only had to deal with one personally - rhodopsin - and it was
a mild case. To minimize dimers/trimers I had to: use sucrose
instead of glycerol in loading buffer, use 200 mM DTT instead
of BME, never ever heat the sample, just dissolve it at RT.
When I deal with NP-40/TX-100 lysates, I add 2X-3X to
loading buffer - these detegents strongly interfere with SDS
Over the years, there's been a number of discussions of
this phenomenon in this group. I have yet to see a coherent
explanation that would explain all things reported. One of them,
mysteries of life :-)
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