Quick navigation:        Home   |    Site Map   ||    References   |    Biography   ||    Copyright   |    Other copyright   |    Contact us   |   
Protein structure
 

Re: [ccp4bb] His tag does not bind.
 

Basic tutorials: 		 
 

CCP4bb navigation

CCP4bb <-- 2007 <-- October 2007 <-- 10 October 2007
Previous message:
Subject: Re: His tag does not bind.
From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2007-10-10 		Next message:
Subject: Re: His tag does not bind.
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-10-11


Subject: Re: His tag does not bind.
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2007-10-10
The presence of a His-tag in the sequence does not automatically mean that
the protein will bind to IMAC resin. Since your protein is so huge and since
the His-MBP-fusion does not work any better than the His-fusion I would
hazard that your protein is highly aggregated in solution, to the extent
that it a) does not enter the beads and b) the His-tags are obscured by all
the other junk.



One of the first things I'd do would be to try detergent lysis of your
culture. This oftentimes can at least reduce the aggregate size down to the
point where they enter the beads.



Artem



_____

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
changrui lu
Sent: Wednesday, October 10, 2007 9:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] His tag does not bind.



Dear all,

I am trying to express a 150 kd protein in E coli. I have it in two
constructs, one with pmal-his and other with only his tag at N terminus. The
full length protein can be detected both by sds and western using anti-his
(190kd and 150kd respectively) but strangely neither binds to his-column
very well. The majority of the full length comes through the column either
at loading step or low salt wash step. The major species that gets trapped
and eluted is the mbp-his truncation (~40kd). Some, though very little, full
length protein did make it out the his column. The pmal-his construct does
not bind amylose resin any better with majority flows right through. All
purification are carried out under standard conditions as mentioned in the
manuals. The protein is soluble and does not precipitate in the columns. I
appreciate and ideas or explanations.

Thanks in advance.

Ray
Cornell Univerisity


CCP4bb navigation

CCP4bb <-- 2007 <-- October 2007 <-- 10 October 2007
Previous message:
Subject: Re: His tag does not bind.
From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2007-10-10 		Next message:
Subject: Re: His tag does not bind.
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-10-11


ProteinCrystallography.org: Copyright 2006-2008 by Quid United Ltd