[ccp4bb] His tag does not bind.

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Subject: atomic FF used in SFALL
From: Bernhard Rupp bernhardrupp {- at -} SBCGLOBAL {- dot -} NET
Date: 2007-10-10

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Subject: Re: His tag does not bind.
From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2007-10-10

Subject: His tag does not bind.
From: changrui lu changrui {- at -} GMAIL {- dot -} COM
Date: 2007-10-10

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: atomic FF used in SFALL
From: Bernhard Rupp bernhardrupp {- at -} SBCGLOBAL {- dot -} NET
Date: 2007-10-10

Next message:
Subject: Re: His tag does not bind.
From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2007-10-10