[phenixbb] need help for crystallization
kevinjin at gmail.com
Fri Mar 30 10:48:49 PDT 2012
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.
On Fri, Mar 30, 2012 at 9:40 AM, Afshan Begum <afshan490 at yahoo.com> 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
> Best Regards
> Dr. Afshan Begum
> University of Hamburg
> Institute of Biochemistry and Molecularbiology
> Laboratory for Structural Biology of Infection and Imflammation
> c/o DESY, Build. 22a
> Notkestr. 85
> 22603 Hamburg
> Home phone: +49 40 22888618
> Fax: +49 40 8998-4747
> E-mail: afshan490 at yahoo.com
Sharing knowledge each other is always very joyful......
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