Lei,
1. Consider making the complex and then purifying the excess peptide
away by dialysis (size exclusion may be tricky since complex may be
diluted in the process).
2. Conventional wisdom would be to try to minimize the amount of excess
peptide as it may "interfere with crystallization". But can I tell you
that if you have 3:1 peptide to protein ratio, excess peptide will
inhibit crystallization? Not really, and in fact quite the opposite
result is possible (won't say likely either way since I don't pretend to
know how to crystallize proteins) - e.g. in some conditions complex may
be disrupted by high salt content and you need excess ligand to keep it
intact. Nobody knows what will work in each particular case - we only
know general trends.
If you have access to a crystallization robot - just try different
ratios. If you don't, consider setting multiple drops on the same
coverslip (with 22mm size four different color thick sharpies taped
together in a rectangular arrangement make a great labeling tool).
Cheers,
Ed.
On Tue, 2010-10-12 at 16:09 -0400, lei feng wrote:
> Hello everyone
>
> first I want to thank you guys in advance. I am trying to
> co-crystallize a 12 KDa protein with a 2 KDa peptide. The biochemistry
> studies showed the binding should be 1:1 . but the binding is not very
> strong ( Kd is around 20-40 uM). I am wondering, is there any
> bad effect of excessive peptide in the solution? someone suggested I
> use lot of peptide , say 3 times of the peptide to protein.
>
> usually what is the ratio you will try when you deal with this
> not-so-small ligand ?
>
> any suggestion is appreciated.
>
> Lei Feng
>
--
"I'd jump in myself, if I weren't so good at whistling."
Julian, King of Lemurs
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