One of the steps that I use in any project which has heavy background is a
change of container prior to unfolding/reduction/alkylation but perhaps you are
already doing this. I think I learned this from technical details reported by
others in doing pull downs but there is no reason it would not be a good
practice for proteomic samples.
D. Eric Anderson
Mass Spectrometry Facility, NIDDK
NIH
Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
(301)-496-7546
Rest of post
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From: turck <email obscured>>
Sent: 24 February 2021 07:24
To: <email obscured>
Subject: [ABRF Discussion Forum] removal of bound media proteins from cell
membrane
The analysis of cellular proteins is often confounded with media proteins, such
as albumin and IgG, that tend to stick to the cellular membrane and end up in
the cellular protein extract. Although we wash several times with PBS we still
find plenty of albumin, IgG and other proteins in our mass spec data.
Can someone recommend methods to avoid isolating media proteins during cell
extraction? Maybe by washing the still intact cells with buffers (high salt?
urea? , ...) that preserve their integrity (no lysis), but remove media
proteins that are bound to the cellular membrane? Or any other method that can
achieve that?
Unfortunately we have very small amounts of cells and cannot afford big losses.
Chris
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