Re: [ccp4bb] mono S vs. mini S vs. S-HyperD

2011-01-27 Thread Ed Pozharski 
Jonas,

scaling up may be tricky as the increased protein concentration affects
the elution profile.  Personally, I tend to think that scaling up may
often be avoided by simply running the protocol that you have developed
several times.  Maximum recommended loading capacity of a 1mL monoS is
25 mg, do you actually need to go much higher? If you can get 10mg of
protein from a single purification, you are in great shape.

As for resolution, the most important factor in my experience is not how
shallow the gradient is (although it matters), but the flow rate.  Some
of them columns have specs that suggest up to 2ml/min flow rates, so
naturally we tend to run them fast (getting 1 column volume per minute
sounds so cool).  But the resolution suffers - I may even go as far as
to say that running the monoQ and such at 1ml/min means you are not
taking advantage of its high resolution at all.  I have seen in the past
high-res ion-exchange columns do miraculous things when you run them at
0.1ml/min.

Perhaps you also want to take a look at UnoS from Biorad.  They are just
as good as monoS (if not better), but are somewhat cheaper.

Good luck,

Ed.

On Tue, 2011-01-25 at 20:06 +, Jonas Boehringer wrote:
> Dear All,
> I am currently looking into various cation exchange resins for the
> separation of mono-, di-, tri-, tetra-,... ubiquitin. So far I have
> been using a small Mono S column but would ideally like to both scale
> up and increase resolution. Before I started searching for this online
> I have never heard of the above resins and was wondering whether
> anybody on this list has any experience, maybe even comparative, with
> anything other than Mono beads (which are really expensive as well).
> 
> 
> Many thanks in advance and best wishes,
> Jonas
> 
> Jonas Boehringer
> Department of Biochemistry
> University of Oxford
> South Parks Rd
> Oxford OX1 3QU
> United Kingdom
> 
> 
> 
> 
>

Re: [ccp4bb] buffers for crystallization

2011-01-27 Thread Eric Larson 

Hi Chandan,

What do you mean by problem in crystallization? Too much precipitate, too many clear drops, ...? If you state the specific problems you are experiencing it is easier to make specific suggestions.  By 388 buffers, do you mean that you have only tried 388 crystallization conditions or ~4 commercial screens? That really is not a lot of conditions to try these days.  Have you tried with and without Zinc?  Are there other known ligands for your protein you can try to use? Have you experimented with other variables such as protein concentration, temperature, ratio of protein solution to reservoir solution, volume of drop, crystallization method (hanging drop vs sitting drop vs. batch vs. ...), etc.  Perhaps the buffer your protein is in going into the crystallization experiments needs to be optimized. Perhaps it is your protein itself that is problem. Do you have a cleavable affinity tag for purification? 
Try crystallizing both the cleaved and non-cleaved protein. You may need to design other constructs of your protein. Move the tag to N- or C-terminus. Make a series of truncation mutants. Maybe you have disordered domains that are preventing crystallization - remove them.  Are there homologous proteins in other species you could try? And the list goes on - there is a near limitless number of things that can be tried.


hope that will at least get you started.

good luck.

Eric


Eric T. Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195

email: larso...@u.washington.edu


On Thu, 27 Jan 2011, chandan kishore wrote:



Hi Everyone,

I have to crystallize one protein which contains a zinc fingure motifs, I am 
facing a problem in crystallization. I already used 388
buffers . Can anyone suggest me some books or papers freely available online on 
Buffers required for crystallization.
Thanks

[ccp4bb] High Throughput crystallisation facility at EMBL Hamburg

2011-01-27 Thread Rosemary Wilson 
The high-throughput crystallization facility at EMBL Hamburg 
 has been available to the 
general user community since 2005. Access to the facility is free of 
charge under the Advanced Infrastructure Initiative P-Cube 
(www.p-cube.eu ), sponsored within the European 
FP7 program. Application requires a brief project description, which is 
evaluated by a transnational access board.


Our platform offers the preparation of initial, optimization and custom 
design crystallization experiments. User have confidential access to the 
results of their experiments, including images, crystallization 
conditions and all relevant experimental parameters under 
http://htx.embl-hamburg.de .


--
Dr. Rosemary Wilson
Scientific Training and Outreach Officer

EMBL c/o DESY,
Building 25a
Notkestrasse 85,
22603 Hamburg,
Germany

Phone: +49 (0)40 89902 216
Fax: +49 (0)40 89902 149
Email: r.wil...@embl-hamburg.de

Re: [ccp4bb] buffers for crystallization

2011-01-27 Thread Prince, D Bryan 
Dear Chandan,

388 buffers are quite a bit. I presume you are talking about intervals of pH 
and not 388 buffers all at one pH. Can you explain a bit more about how you 
determined that the buffer is the problem, and what results led you to that 
conclusion?

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of chandan 
kishore
Sent: Thursday, January 27, 2011 1:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] buffers for crystallization


Hi Everyone,

I have to crystallize one protein which contains a zinc fingure motifs, I am 
facing a problem in crystallization. I already used 388 buffers . Can anyone 
suggest me some books or papers freely available online on Buffers required for 
crystallization.
Thanks


--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.
 

[ccp4bb] Interdisciplinary Postdoc position available et EMBL

2011-01-27 Thread Panne Daniel 
A postdoc position at the interface of structural biology and high throughput 
genome analysis is available at the EMBL. The position is part of the EMBL 
interdisciplinary postdoc programme and the accepted candidate will work in 
close collaboration with the Furlong Group in Heidelberg, Germany and the Panne 
Group in Grenoble, France. It is also possible to design and propose an 
interdisciplinary project according to one's own scientific interest. 
Positions are initially funded for 3 years. Application deadline is 20th March.
EMBL is an exciting, international, dynamic place to work, and is equipped with 
a state of the art facilities. The Grenoble laboratory is located on an 
international research campus providing one of the best environments for 
structural biology in Europe.
Further details are available here:
http://www.embl.de/training/postdocs/eipod/index.html

-- 
Daniel Panne, PhD
EMBL
Structure and Function of Macromolecular Assemblies
pa...@embl.fr