Inhibition of DTT

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri May 5 15:26:51 EST 2000


In article <8esucf$htp$1 at pegasus.csx.cam.ac.uk>, "Choe Woo-seok" <wsc22 at cam.ac.uk> wrote:
:
:I'm going to purify one of His-tag proteins produced as an inclusion body in
:E. coli.
:I have to use Dithiothreiotol (DTT) or any other reducing agent to break
:disulfide bridges
:of my target protein to solubilize it.  I applied the whole cell lysate
:including
:the target protein onto the Ni-NTA and Ni-IDA resins to test the efficiency
:of affinity binding
:of my protein with those resins.  Due to the presence of DTT (20 mM), the
:nickel ion seemed
:to be reduced and I could not find my target protein in the eluted
:fractions.
:
:Although one of the simplest way to remove DTT before applying the cell
:lysates onto affinity
:resins might be the diafiltration or gel permeation chromatography, I would
:rather prefer avoid
:this DTT removal step.
:
:Are there any methods or agents to inhibit reducing reaction by DTT without
:affecting the proteins?
:

If you must use DTT then you don't have much choice. I would use 100 mM
beta ME in place of 20 mM DTT during solubilisation step, then would
dilute 5X to reduce bME concentration to 20 mM which Ni-NTA tolerates 
more or less OK (even with this, you'll probably need to strip and 
recharge the column with new Ni2+ after every use). 

        - Dima





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