Dear Careina,
First: make sure your solution is completely free of competing primary amines,
e.g. tris buffer. Phosphate buffers or HEPES should be used; if you need buffer
exchange, do it by gel filtration or extensive dialysis (2-3 changes), not
concentration-dilution.
Second: Try to use BS3,
Dear CCP4 members
I do not have any good experience with chemical cross linking, it has not
worked for me and I am curious how others have got it to work. In my latest
attempt I wished to crosslink two monomers with DSS. The protein is exclusively
dimeric according to SEC. I incubated the prote
Thanks all for the comments. I was thinking of crosslinking a protein that
hasn't crystallized. Cystein engineering seems a good idea but depends on the
availability of a good model. We'll be trying mild crosslinking using
bifunctional reagents of various lengths (I suspect glutaraldehyde will n
Hi Opher,
I have a project where a protein was made 'cysless' and selective cysteines
were introduced to allow for directed crosslinking using MTS and aldrithiol
activated reagents (see Toronto Research Company and Pierce for a range of
reagents of different lengths). A side product of these cr
n board wrote: -
To: CCP4BB@JISCMAIL.AC.UK
From: Boaz Shaanan
Sent by: CCP4 bulletin board
Date: 08/15/2011 10:11AM
Subject: Re: [ccp4bb] Protein crosslinking before crystallization
Hi Opher,
I seem to recall some old papers in which glutarldehyde was used (in the
RESERVOIR only) in or
CP4BB@JISCMAIL.AC.UK] on behalf of Opher Gileadi
[opher.gile...@sgc.ox.ac.uk]
Sent: Monday, August 15, 2011 5:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein crosslinking before crystallization
Hi all
Does anyone have experience or insights on intramolecular crosslinking of
proteins b
Hi all
Does anyone have experience or insights on intramolecular crosslinking of
proteins before crystallization? The idea is to lock a felxible (multidomain)
protein into a restricted conformation.
Thanks,
Opher
