Inhibition of DTT

Blitzkrieg tyson at canada.com
Fri May 5 21:21:06 EST 2000


..wouldn't go higher than 10mM DTT
...if possible you can try using B-ME which will work will with the Ni-NTA
column up to 50mM




"Dima Klenchin" <klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu> wrote in
message news:8evasn$be8$3 at news.doit.wisc.edu...
> In article <8esucf$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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