Insect supernatant inducing color change in Talon resin?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Thu Feb 22 08:58:39 EST 2001


vladimir vigdorovich <vladv at u.washington.edu> wrote:
:Hi, I am trying to optimize a purification procedure of a His-tagged
:protein derived from Hi5 cells using Talon resin. When I apply clarified,
:pH-adjusted (to ~8.0), buffered (supplemented with 20 mM Tris-HCl) supe to
:the column, it slowly changes color to whitish-pink and the flowthrough
:darkens. Some protein binds but I suspect that much ends up in
:flowthrough.

Can't you run a gel and see if you indeed have significant amounts
of protein in flow through? 

:Can anyone verify my suspicions that Co2+ is being leeched and/or suggest
:a remedy?

I do Ni-NTA on insect extracts routinely, and have not seen color 
changes. I am not sure but perhaps Co2+ is reduced easier
than Ni2+. Tris can reduce metals, albeit very inefficiently. The
higher is pH, the faster reduction goes. If the problem is 
reduction, you can try pH 7.0 buffered with phosphate or
try charging column with Ni2+. With NTA, leeching is practically
excluded, but Talon is not NTA and I don't think they tell
what exactly it is (if IDA then it can happen - IDA chelates a lot 
weaker). 

        - Dima






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