Hello,
When doing affinity pulldown experiments destined for MS analysis using either
antibodies or bait proteins I and others usually see results that look more
like a whole cell extract rather than a subset of enriched proteins on a
background of lower intensity cell proteins. The non-specific binding
background of proteins seems to be intractable.
Has anyone developed a protocol which can overcome the high non-specific
binding in AP-MS experiments? I am fairly familiar with relevant literature,
but nothing seems to work will for us.
Best regards,
Brian
Hi Brian,
Probably something you are already doing, but transferring the beads with the
bound sample to a new Eppendorf tube is a key step to include in order to
eliminate non-specific binding on the plasticwareโฆ
Other things to consider are a gentle detergent-containing wash, followed by a
few non-detergent buffer rinses prior to elution of the bound proteins. It is a
balance between losing the truly, but weakly bound proteins and getting rid of
the truly adventitiously bound stuff.
Best of luck!
Martin
Martin Middleditch
Lead Technologist (Mass Spectrometry)
School of Biological Sciences
University of Auckland
Room 301-422
23 Symonds Street
Auckland
(09) 923-7612 or 923-7540
From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian
Sent: Thursday, 18 November 2021 12:23 pm
To: <email obscured>
Subject: [ABRF Discussion Forum] AP-MS pull down question
Hello,
When doing affinity pulldown experiments destined for MS analysis using either
antibodies or bait proteins I and others usually see results that look more
like a whole cell extract rather than a subset of enriched proteins on a
background of lower intensity cell proteins. The non-specific binding
background of proteins seems to be intractable.
Has anyone developed a protocol which can overcome the high non-specific
binding in AP-MS experiments? I am fairly familiar with relevant literature,
but nothing seems to work will for us.
Best regards,
Brian
โโ
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