1 imidazole affinity is not high which is why you use 200 mM or more to
elute. So it comes off by itself.
2 you can wash with low ph and then recharge this is somewhat easier on the
resin
On Sep 15, 2011 9:25 PM, "m zhang" <mzhang...@hotmail.com> wrote:
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> Dear all,
> I have two questions:
> First, I was trying to crystallize a complex of two proteins. Both
proteins has been crystallized before. The two proteins bind to each other
based on Biacore study, but they didn't form a single peak on gel
filtration. When I mixed them at 1:1 ratio, the crystals I got contain only
one of the two proteins. I was suggested to increase the ratio, for example
1.5:1, to increase the probability of co-crystallization which I will try.
But I do want to hear if there are other possible ways to try. What would
you try if you were in my situation?
> Second is about reusing of Ni-NTA resin. According to Qiagen's
instruction, after using fresh Ni-NTA resin, one only needs to wash the used
Ni resin first with 0.5M NaOH, then with your own buffer. After that the
resin is ready to be reused until it needs being recharged. But my question
is: Once immidazole competes with His-tagged protein and binds to Ni-resin,
how can immidazole be rinsed off with the same buffer(usually pH is above 7)
one uses to purify the protein?
> Thank you for any suggestion or comment.
> Min
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