need help for crystallization

From: Kevin Jin
Date: 30 March 2012 18:48


My pleasure !
Since this may be help for other people, I  also cc it to CCP4BBS.

According to your email, I guess the buffer is phosphate buffer at pH
7.4.  You can do a quick buffer exchange before crystallization.

Since PO4 is a competitor in this case, I will also avoid PO4 and
Cocodylate buffer from screen kits.

Here is what you can do,
1. concentrate your protein using an amicro centrocon with 3KD cutoff.
2. Measure how much buffer has been spin down, then add equal amount
of Tris with same pH back to the top.
3. Then, spin down again.
4. Repeat several times, 5 times may be good enough.
5. Most of Phosphate buffer should be removed.

That's what I used before.

Let me know if it works.

Best,

Kevin Jin





On Fri, Mar 30, 2012 at 9:40 AM, Afshan Begum > wrote:
>
>  Dear Kevin
>
> I have seen your website and come to you to discuss my problem actually i
> want to co-crystallized my enzyme with inhibitor but problem is that the
> phosphate ion come to the active site and occupied the cavity and my
> inhibitor not bind to the active center i purchase this enzyme from a
> commercial source and they purify  with phosphate buffer that's why also
> phosphate is occupied the cavity how can i get ride of it from the active
> center could you kindly help me regarding this i would be really thankful to
> you.
>
>
> Best Regards
>
> AFSHAN
> ===========================================
> Dr. Afshan Begum
>



--
Kevin Jin

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Website: http://www.jinkai.org/