Re: [ccp4bb] How to replace 2.45M K/Na phosphate buffer - 2

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: How to replace 2.45M K/Na phosphate buffer - 2
From: Claudia Scotti claudiascotti {- at -} HOTMAIL {- dot -} COM
Date: 2007-06-22

Next message:
Subject: Re: How to replace 2.45M K/Na phosphate buffer - 2
From: Kornelius Zeth kornelius {- dot -} zeth {- at -} TUEBINGEN {- dot -} MPG {- dot -} DE
Date: 2007-06-22

Subject: Re: How to replace 2.45M K/Na phosphate buffer - 2
From: "Murray, James W" j {- dot -} w {- dot -} murray {- at -} IMPERIAL {- dot -} AC {- dot -} UK
Date: 2007-06-22

Dear Claudia,

>These crystals dissolve in several cryoprotectant solutions and survive only in malonic acid, with which they give diffraction at 7-8 A.

You do not mention what the room temperature diffraction is like. If you have not tested it already, you should do so - even without malonate, you might only get 7-8A diffraction. Assuming the room-temp diffraction is OK, you could just try increasing the concentration of phosphate buffer - high salt can also act as a cryoprotectant.

James

Dr. James Murray
Biochemistry Building
Department of Biological Sciences
Imperial College London
London, SW7 2AZ
Tel: +44 (0)20 7594 5276

-----Original Message-----
From: CCP4 bulletin board on behalf of Claudia Scotti
Sent: Fri 22/06/2007 13:42
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] How to replace 2.45M K/Na phosphate buffer - 2

Sorry: forgotten the pH: the crystals grow between pH 6.5 and 6.7.

Claudia

Claudia Scotti
Dipartimento di Medicina Sperimentale
Sezione di Patologia Generale
Universita' di Pavia
Piazza Botta, 10
27100 Pavia
Italia
Tel. 0039 0382 986335/8/1
Facs 0039 0382 303673

----------------------------------------
> Date: Fri, 22 Jun 2007 14:15:47 +0200
> From: claudiascotti@HOTMAIL.COM
> Subject: [ccp4bb] How to replace 2.45M K/Na phosphate buffer
> To: CCP4BB@JISCMAIL.AC.UK
>
> Dear list,
>
> I've crystals in a condition including 2,45 M K/Na phosphate buffer for the well condition, and 1 microl protein + 0.8 microl Na/K phosphate buffer + 0.25 microl 30% 1,6-hexanediol in the drop condition.
>
> These crystals dissolve in several cryoprotectant solutions and survive only in malonic acid, with which they give diffraction at 7-8 A.
>
> Among the several different possibilities, I'd like to try to optimize the crystals, and one possiblity among others would be to change the buffer.
>
> Any suggestions, please, about how to replace K/Na phosphate? At these concentrations it is acting as a buffer but also as a precipitant and I was wondering if anybody has experienced nice buffer-salt combinations that proved to be useful starting from a similar condition.
>
> Thanks a lot,
>
> Claudia
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> Claudia Scotti
> Dipartimento di Medicina Sperimentale
> Sezione di Patologia Generale
> Universita' di Pavia
> Piazza Botta, 10
> 27100 Pavia
> Italia
> Tel. 0039 0382 986335/8/1
> Facs 0039 0382 303673
> _________________________________________________________________
> Make every IM count. Download Windows Live Messenger and join the i'm Initiative now. It's free.
> http://im.live.com/messenger/im/home/?source=TAGWL_June07

_________________________________________________________________
Make every IM count. Download Windows Live Messenger and join the i'm Initiative now. It's free.
http://im.live.com/messenger/im/home/?source=TAGWL_June07

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: How to replace 2.45M K/Na phosphate buffer - 2
From: Claudia Scotti claudiascotti {- at -} HOTMAIL {- dot -} COM
Date: 2007-06-22

Next message:
Subject: Re: How to replace 2.45M K/Na phosphate buffer - 2
From: Kornelius Zeth kornelius {- dot -} zeth {- at -} TUEBINGEN {- dot -} MPG {- dot -} DE
Date: 2007-06-22