[ccp4bb] HydrophobiC interaction Chromo

[Meg]

Dear all,

In continuation with my earlier mail on problem in cation exchange chromato, i 
was suggested to go for hydrobic interaction chromato [HIC] as an additional 
step. my protein is indeed a hydrophobic protein. we have sepharose 6 fast 
flow low sub resin and my protein has a pi of 5.8 -6.3. one of the patent 
papres relating to the same moleculte it was mentioned HIC in suitable buffer 
containing 0.5 M NH4SO4 pH of 5.0 and elution in same buffer without NH4SO4 
pH 5.0. They have mentioned addition of 2-20% ethanol in the elution buffer. 
is it O.K. to add ethanol in buffer.

Can anyone provide me a suitable buffer recipe to use at pH 5.0 for HIC.

i performed the HIC but the resolution was not good and my sample was 
diluted 10 fold and elution was near end of elution gradient.

Kindly comment

thanks in anticipation

[ccp4bb] TLS parameter representation

[cedric bauvois]

Dear All,

i would like to know wether it's possible to represent graphically
TLS-tensors obtained via refmac5 and how to procede?

Best Regards.

Cédric

-- 
Dr. Cedric Bauvois
Cristallographie des protéines
Institut de Recherches Microbiologiques JM Wiame -IRMW
Av E. Gryzon 1, 1070 Brussels (Belgium)
tél: +32 (0)2 5273634
fax: +32 (0)2 5267273

Re: [ccp4bb] HydrophobiC interaction Chromo

[David Briggs]

Hi Meg,

ethanol is often used in a HIC elution buffer as it is good at
breaking hydrophobic/hydrophoic interactions. (Detergents or
isopropanol can be used to do the same thing). But, as with all
things, tolerance to ethanol depends upon your protein.

At pH 5, I would guess that Citrate or Acetate buffers are most useful.
See http://www.liv.ac.uk/buffers/ for buffer recipes.

As for dilution of your sample, optimisation of the gradient you use
can reduce the volume your peak elutes in.

Regards,

David

2008/9/26 Meg <[EMAIL PROTECTED]>:
> Dear all,
>
> In continuation with my earlier mail on problem in cation exchange chromato, i
> was suggested to go for hydrobic interaction chromato [HIC] as an additional
> step. my protein is indeed a hydrophobic protein. we have sepharose 6 fast
> flow low sub resin and my protein has a pi of 5.8 -6.3. one of the patent
> papres relating to the same moleculte it was mentioned HIC in suitable buffer
> containing 0.5 M NH4SO4 pH of 5.0 and elution in same buffer without NH4SO4
> pH 5.0. They have mentioned addition of 2-20% ethanol in the elution buffer.
> is it O.K. to add ethanol in buffer.
>
> Can anyone provide me a suitable buffer recipe to use at pH 5.0 for HIC.
>
> i performed the HIC but the resolution was not good and my sample was
> diluted 10 fold and elution was near end of elution gradient.
>
> Kindly comment
>
> thanks in anticipation
>



-- 

David C. Briggs PhD
Father & Crystallographer
http://drdavidcbriggs.googlepages.com/home
AIM ID: dbassophile

Re: [ccp4bb] TLS parameter representation

[Martyn Winn]

The AXES keyword in tlsanl allows import and display in CCP4mg or
MOLSCRIPT.

TLSView from Jay Painter and Ethan Merritt can also do it.
http://pymmlib.sourceforge.net/

Cheers
Martyn

On Fri, 2008-09-26 at 10:08 +0200, cedric bauvois wrote:
> Dear All,
> 
> i would like to know wether it's possible to represent graphically
> TLS-tensors obtained via refmac5 and how to procede?
> 
> Best Regards.
> 
> Cédric
> 
> -- 
> Dr. Cedric Bauvois
> Cristallographie des protéines
> Institut de Recherches Microbiologiques JM Wiame -IRMW
> Av E. Gryzon 1, 1070 Brussels (Belgium)  
> tél: +32 (0)2 5273634
> fax: +32 (0)2 5267273
> 
-- 
***
* *
*   Dr. Martyn Winn   *
* *
*   STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K.   *
*   Tel: +44 1925 603455E-mail: [EMAIL PROTECTED]*
*   Fax: +44 1925 603825Skype name: martyn.winn   * 
* URL: http://www.ccp4.ac.uk/martyn/  *
***

Re: [ccp4bb] TLS parameter representation

[abhik mukhopadhyay]

Hi Cedric
TLSVIEW does that one. You will get the software from the web site  
http://pymmlib.sourceforge.net/tlsview/tlsview.html.


Regards
Abhik

Re: [ccp4bb] Reading the old literature / truncate / refinement programs

[Ian Tickle]

This goes back to my previous idea about using the Bayesian estimates
( & sig()) of I & sig(I) in the refinement instead of the measured
ones.  This would remove any objection to using negative observed
intensities, though it's hard to see what exactly the objection is.
Basically you're just moving the threshold for the now deprecated
practice of applying a sigma cutoff down from 3 (or whatever) to zero,
and the same objections to this practice still apply, as you point out.

The difference between using Imeas and  would in practice only be
that you would get different R factors for the weak data (i.e.
definitely lower and maybe even more realistic, so you might be in a
better position to judge the true worth of the weak data).  As I said
before it's debatable that it would give you a better refined structure,
since you are not putting in any new information, as compared with
simply using all the Imeas data, including the negative ones.

I suggested before that there would only be an effect on the outcome as
a result of using the Bayesian estimate of I if the intensities
themselves were the focus of the experiment, and I said that this is not
usually the case.  However 'usually' is the key word here: there are
situations where the intensities are the focus of interest, namely in
testing for twinning.  My understanding is that currently what happens
is that to obtain the intensity values needed for the twinning tests,
the Bayesian estimates  are simply squared.  Of course you could also
use Imeas but the Bayesian estimate ought to work much better,
particularly for the Britton and related tests which rely on detecting
negative intensities on detwinning.  The problem with using ^2 is of
course that it's not equal to  (particularly for weak data) so
there's a strong argument that that procedure is bad statistics.  In
practice of course the weak data is often omitted for the test because
it's unreliable, but then maybe the reason it's unreliable is that it's
incorrectly calculated!  I haven't tried testing this yet, mainly
because I don't yet have a prog that will compute the  values in MTZ
format.

Cheers

-- Ian

> -Original Message-
> From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED]
On
> Behalf Of Dunten, Pete W.
> Sent: 26 September 2008 00:01
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Reading the old literature / truncate / refinement programs
> 
> 
> 
> I mentioned previously phenix.refine tosses your weak data if IMEAN,
> SIGIMEAN are chosen during refinement.
> 
> 
> 
> I'm wondering if this omission of weak Fobs from the Fobs-Fcalc
difference
> map explains why the difference maps out of refmac seem to be more
helpful
> in showing where to move atoms.
> 
> 
> 
> D. Crowfoot et al. in The Chemistry of Penicillin (1949) explain why
this
> might be so, and Stout & Jenson elaborate the argument.  Briefly, the
> calculated phase will be closest to the phase of the vector difference
> Fcalc - Fobs when |Fcalc| > |Fobs|.
> 
> 
> 
> I leave it to the reader to try calculating some maps with and without
the
> weak Fobs in phenix.refine or refmac, and perhaps making some
deliberate
> rotamer errors, to see if using the complete data with weak Fobs
helps.



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Re: [ccp4bb] losing zinc during crystallization

[Fischmann, Thierry]

Loss of a zinc at a dimer interface, triggered by formation of a disulfide 
bridge across the interface has already been observed for nitric oxide 
synthase. The 1st reported structure had the disulfide bridge:
http://www.sciencemag.org/cgi/content/abstract/279/5359/2121

But 2 other groups independently found a structural zinc coordinated by 4 cys, 
2 from each monomer:
http://www.cell.com/content/article/abstract?uid=PIIS0092867400817183
and:
http://www.nature.com/nsmb/journal/v6/n3/abs/nsb0399_233.html

Since the protein still has the zinc prior to the crystallization trial, I 
suggest to try to limit as much as possible the amount of oxygen in your 
buffer. You could degas the precipitant (preferentially at room temperature), 
then bubble an inert gas (nitrogen is cheap ...) in (preferentially in the 
cold, even if you need to let the buffer re-equilibrate at the temperature of 
your crystallization conditions. The rationale for using two temperatures is 
that gas solubility decreases with temperature). The trail can also be moved in 
a small plastic bag connected to a nitrogen line, and punctured with a small 
hole to maintain a constant nitrogen flow. This very cheap set-up works very 
well as an economical anaerobic chamber (we've had success with an 
oxygen-sensitive protein using this set-up). The only oxygen left will be in 
the air initially trapped between the cover slip and the well solution. But 
most plastics let oxygen diffuse through: as long as you have enough initial 
reducing agent to protect the protein for ~ 24 hrs, the above should work.

Good luck
Thierry

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Engin Ozkan
Sent: Thursday, September 25, 2008 06:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] losing zinc during crystallization

Here is my two cents...

How strong zinc is captured by the protein is very protein dependent: I 
always thought that a great case for this variability was the protection 
of RING ubiquitin ligases against NEM. Cysteine-catalyzing HECT 
ubiquitin ligase are killed by NEM; RING finger ligases, where zinc is 
assumed to be only structural, are thought to be NEM resistant... Well, 
until you actually do the experiment and see that some RING ligases are 
also inactivated by NEM! My theory was that I had a case of weak zinc 
coordination, where oxidation of the zinc-coordinating cysteines was a 
big issue. DTT made it worse, TCEP kept the protein unaggregated the 
longest.

That's just one protein, though.

Engin

Artem Evdokimov wrote:
>
> Please note that TCEP decomposes and one of the decomposition products 
> is phosphate. Enough TCEP and you might have Zinc Phosphate crystals 
> which can sometimes look very odd and 'protein looking'.
>
> Artem
>
> 
>
> *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf 
> Of *Jennifer Han-Chun Tsai
> *Sent:* Thursday, September 25, 2008 6:18 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] losing zinc during crystallization
>
> I don't know if anyone had experience of TCEP inducing zinc acetate to 
> precipitate. This paper mentioned this.
>
> The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution
>
> *Paul C. Smith, Stuart Firestein and John F. Hunt*
>
> *Journal of Molecular Biology 
> 
> Volume 319, Issue 3 
> ,
>  
> 7 June 2002, Pages 807-821 *
>
> I saw if protein sample containing TCEP would either form 
> precipitation or weird and rounded shape of small molecule crystal but 
> not salt. I have tried to crush that rounded shape crystal. It's quite 
> soft, very similar to the way protein crystals got crushed.
>
> Does anyone have this experience? Or actually this kind precipitation 
> could get nice crystal after optimization?
>
> Thanks,
>
> Jennifer Tsai
>
> On Thu, Sep 25, 2008 at 4:53 PM, Eric <[EMAIL PROTECTED] 
> > wrote:
>
> We had a zinc-finger containing protein that we were soaking with 
> different heavy atom compounds. It turns out KAu(CN)2 provided the 
> best diffraction of several soaks. We found out it was because the 
> gold had replaced the zinc ion and was coordinated by the Cys and 
> His's. The lab nickednamed the protein Goldfinger! Although we didn't 
> have the problem with disulfides, perhaps a similar gold soak might 
> help if you tried to crystallize the protein in the presence of TCEP 
> as well.
>
> HTH
>
> - Original Message - From: "Sue Roberts" 
> <[EMAIL PROTECTED] >
> To: mailto:CCP4BB@JISCMAIL.AC.UK>>
> Sent: Thursday, September 25, 2008 4:35 PM
> Sub

[ccp4bb] deadline for applications for beamtime at BM14 -RUN oct-DEC 2008

[Martin A. Walsh]

Dear all, deadline for submission of proposals for beamtime at BM14 for
beamtime at ESRF during Nov/Dec 2008 is next Friday 3rd october 2008 -get in
your proposals now!. Beamline proposals can be submitted on-line from the
BM14 webpages at http://www.bm14.ac.uk    

PLEASE remember this call is distinct from ESRF proposal call and allows you
direct access to BM14 -special requests can be forwarded directly to me and
we will do our best to accommodate you.

BEAMLINE FEATURES

**MARMOSIAC 225 CCD detector  (

http://www.mar-usa.com/products/mx_series.htm)

**MD2 Microdiffractometer
(http://www.embl-grenoble.fr/groups/instr/MD2-17.pdf) 

**MiniKappa goniostat (http://www.embl-grenoble.fr/groups/instr/MK2.pdf
-videos available here
-http://www.embl-grenoble.fr/groups/instr/mk2videos.html)

**Robotic Sample changer (

http://journals.iucr.org/d/issues/2006/10/00/gx5085/index.html)

**Easily Tuneable over the 6.5 - 18 keV range with X-ray flux at sample
through 100micron aperture  of ~1.5x10*10 photons/s/200mA beam current at
12.7keV 

**REMOTE ACCESS for data collection 

FULL CALL DETAILS follow:




SYNCHROTRON BEAM TIME FOR MACROMOLECULAR CRYSTALLOGRAPHY AT THE UK/EMBL MAD
BEAMLINE BM14, ESRF 

Scheduling Period: Nov/Dec 2008

DEADLINE for proposals: 3rd October 2008


-

Call for proposals from EMBL member states, EU states and associated states
for Nov/Dec 2008 synchrotron run at ESRF. The bending magnet MAD beamline
BM14 at the ESRF is being operated as a UK Collaborative Research Group(CRG)
beamline in collaboration with the EMBL Grenoble Outstation. Proposals for
beam time can be made via a user-friendly web based application form, both
for monochromatic and multi-wavelength experiments.

Beam time is available to groups from the UK and EMBL, EU and EU associated
member states. Deadline(25th July 2008) for beamtime proposals at BM14/ESRF
for Nov/Dec 2008 

Reimbursement is available for travel and subsistence for groups based in
the UK based and EU member or associate member states (see NeedToKnow Link
in Main menu on Beamline Homepage for details on reimbursement and other
useful information)

Full details of the beamline characteristics, the user program and the
procedure for the submission of electronic proposals can be found at the
beamline homepage http://www.bm14.ac.uk   or by
following the links from  
http://ww.embl-grenoble.fr or   http://www.esrf.eu 


--

Please don't hesistate to contact me if you have any queries

Thanks

Martin

 

 

-

Martin Walsh,

MRC Group Leader and BM14 Responsible

Medical Research Council (MRC) France,

CRG BM14,

c/o ESRF,

6, rue Jules Horowitz,

B.P. 220

38043 Grenoble CEDEX

France

Tel:   +33 4 38.88.19.69

Fax:  +33 4 76.88.23.80

email1:   [EMAIL PROTECTED]

email2:   [EMAIL PROTECTED]

 

 

 

[ccp4bb] Research Associate (Staff Scientist ? Structural Biology & X-ray Science)

[Winter, G (Graeme)]

On behalf of Monica Wesley:

Research Associate (Staff Scientist – Structural Biology & X-ray Science)

Cornell High Energy Synchrotron Source

?The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron 
Source (MacCHESS) is seeking to fill a staff scientist position. Applicants 
should have a Ph.D. degree in a relevant area of science, as well as relevant 
postdoctoral experience. Those with experience in x-ray solution scattering on 
biological systems (SAXS and WAXS), macromolecular crystallographic phasing 
methods, or high-pressure protein crystallography are encouraged to apply. Of 
special interest are applicants who can take structural biology into the future 
with the coherent, high-spectral brilliance, time-resolved x-rays expected from 
next-generation x-ray sources such as Cornell's Energy Recovery Linac (ERL) 

??Located on an ivy-league university campus in picturesque upstate New York, 
the Cornell High-Energy Synchrotron Source (CHESS) serves a world-wide user 
base of structural biologists, chemists, physicists, and engineers. MacCHESS is 
an NIH-supported National Resource providing support for structural biology at 
CHESS. Staff scientists are expected to initiate and carry out innovative 
research leading to advances in the application of X-radiation to key 
challenges in structural biology. In addition, scientists provide support to 
visiting research groups, collaborate with leading researchers at other 
institutions, and disseminate information to the scientific community. MacCHESS 
is a heavily team-oriented environment. Good communication skills, including 
fluency in the English language, and the ability to work in a group, are 
necessary. ??

Qualified candidates should send their curriculum vitae and arrange for three 
letters of recommendation to be sent to Kathy Dedrick (kd73 at cornell dot edu) 
200L Wilson Lab, Ithaca, NY 14853 

??Deadline for application is November 1, 2008. Starting date is negotiable. 
Cornell is an equal opportunity employer.



Research Associate MacCHESS.doc
Description: Research Associate MacCHESS.doc

Re: [ccp4bb] Off-topic: coomassie linearity? SUMMARY

[Jacob Keller]

Hi All,

thanks for the responses. It seems that coomassie linearity is not so bad 
after all, especially when measured by IR fluorescence (reference from Steve 
Darnell). Others mentioned silver stain, but it seems that silver has a 
narrower dynamic range, even though more sensitive. Sypro and Biorad 
flamingo seem linear, and as sensitive as silver. But...for the fluorescent 
dyes, one optimally needs a fluorescence scanner.


Thanks all,

Jacob Keller

ps The original coomassie reference seems to be

S. Fazekas de St. Groth, R.G. Webster and A. Datyner, Two new staining 
procedures for quantitative estimation of proteins on electrophoretic 
strips, Biochim. Biophys. Acta 71 (1963), pp. 377-391.


***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***

- Original Message - 
From: "Gregory Alushin" <[EMAIL PROTECTED]>

To: 
Sent: Thursday, September 25, 2008 6:07 PM
Subject: Re: [ccp4bb] Off-topic: coomassie linearity?


The Bio-Rad Flamingo Fluorescent Gel Stain has a very linear profile  with 
protein concentration (if you believe the standard curves in the  manual). 
In my hands it gives nice results for binding assays.


Cheers,
-Greg Alushin

On Sep 25, 2008, at 2:35 PM, Jacob Keller wrote:


Dear Crystallographers,

I remember having seen on this listserve that coomassie stain is 
horribly non-linear in intensity per protein concentration, which  leads 
me to two questions:


1. Does anybody have a reference for quantitation of coomassie's 
linearity (and possibly other stains), i.e. where and how extensive  the 
linear range is, and

2. Can anybody suggest a more linear protein gel stain?

Thanks,

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***

Re: [ccp4bb] HydrophobiC interaction Chromo

[Artem Evdokimov]

Dear Meg,

Please note that there are many ways to do HIC. Different resins result in
different degrees of separation and it's unfortunately impossible to predict
what would happen without trying it out. Which is why various companies sell
HIC 'trial kits' - a few little columns packed with media substituted with
various chains - so that you can try on a small scale. Hopefully you can lay
your hands on one of those kits or on something similar.

As others pointed out, HIC elution peaks are not normally sharp - this is
expected given the nature of the interaction between the matrix and the
protein. The resolution issue is either a) due to incompatible resin b)
because even your 'good' protein might be not entirely folded after all or
c) because you've not found the right kind of salt/additive combination to
do the HIC om.

Notably, pH plays a role as well. If as I pointed out earlier, your protein
survives transition from low to high pH this might open doors to other
purification venues.

As far as buffers for pH 5.0 - Citrate or MES should work, IMHO.

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg
Sent: Friday, September 26, 2008 4:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] HydrophobiC interaction Chromo

Dear all,

In continuation with my earlier mail on problem in cation exchange chromato,
i 
was suggested to go for hydrobic interaction chromato [HIC] as an additional

step. my protein is indeed a hydrophobic protein. we have sepharose 6 fast 
flow low sub resin and my protein has a pi of 5.8 -6.3. one of the patent 
papres relating to the same moleculte it was mentioned HIC in suitable
buffer 
containing 0.5 M NH4SO4 pH of 5.0 and elution in same buffer without NH4SO4 
pH 5.0. They have mentioned addition of 2-20% ethanol in the elution buffer.

is it O.K. to add ethanol in buffer.

Can anyone provide me a suitable buffer recipe to use at pH 5.0 for HIC.

i performed the HIC but the resolution was not good and my sample was 
diluted 10 fold and elution was near end of elution gradient.

Kindly comment

thanks in anticipation

Re: [ccp4bb] losing zinc during crystallization

[Guenter Fritz]

Hi Sue,
I fully agree with Thierry, you might have to cyrstallize the protein 
under exclusion of dioxygen. There are many metallo proteins which have 
to be crystallized that way. But a first attempt might be the TCEP as 
already suggested by many others. If you need advice regarding 
crystallization without O2, send me a note.

Good luck!
Guenter

Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what 
seems to me to be an eternity. The protein contains stoichiometric 
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required 
for activity.  Each crystal we've obtained has lost the zinc and 
contains a disulfide bond between two cysteine residues that should be 
zinc ligands (based on structures of similar proteins).


We've tried crystalizing in the presence of reducing agents, 
crystallizing with substrate analogs, and supplementing the 
crystallization drops with zinc with no success (and combinations of 
these approaches).  We've obtained a variety of crystals and 
determined structures, but none contain any zinc.


Attempts to insert zinc into the crystal (zinc + reducting agent or 
zinc alone) have not been successful.


Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]

[ccp4bb] FACULTY POSITION IN X-RAY CRYSTALLOGRAPHY

[John Pascal]

FACULTY POSITION IN X-RAY CRYSTALLOGRAPHY

Where:
Department of Biochemistry and Molecular Biology
Thomas Jefferson University in Philadelphia USA


Job Description:
The Department of Biochemistry and Molecular Biology at Thomas Jefferson
University in Philadelphia invites applications for tenure-track or tenured
faculty positions in the areas of X-ray crystallography of proteins or nucleic
acids. We seek outstanding established investigators with demonstrated research
excellence and a solid track record of extramural funding. The Department
offers a highly collaborative culture and provides state-of-the-art facilities
for advanced structural biology and biophysical work. The successful candidate
is expected to establish a dynamic and independently funded research program
and participate in graduate training at the interface between biology,
biochemistry, and biophysics.


How to respond:
Applicants should submit a curriculum vitae, a brief statement of research
interests and future plans, and names of at least three references to:
Professor Ya-Ming Hou, Thomas Jefferson University, Department of Biochemistry
and Molecular Biology, 233 South 10th Street, BLSB 220, Philadelphia, PA 19107.
Email: [EMAIL PROTECTED]

Thomas Jefferson University is located in center city Philadelphia, adjacent to
a variety of cultural, entertainment and historical attractions.  Affirmative
Action/Equal Opportunity Employer.


John Pascal, PhD
Thomas Jefferson University
Department of Biochemistry & Molecular Biology
233 South 10th Street, BLSB 804
Philadelphia, Pennsylvania  19107

ph 215.503.4596
fx  215.923.2117

Re: [ccp4bb] Off-topic: coomassie linearity? SUMMARY

[Juergen Bosch]

On 26 Sep 2008, at 08:20, Jacob Keller wrote:


Hi All,

thanks for the responses. It seems that coomassie linearity is not  
so bad after all, especially when measured by IR fluorescence  
(reference from Steve Darnell). Others mentioned silver stain, but  
it seems that silver has a narrower dynamic range, even though more  
sensitive. Sypro and Biorad flamingo seem linear, and as sensitive  
as silver. But...for the fluorescent dyes, one optimally needs a  
fluorescence scanner.
You can visualize Sypro Orange on a regular Ethidium bromide detection  
system, but a computer based detection system is more sensitive.


Jürgen




Thanks all,

Jacob Keller

ps The original coomassie reference seems to be

S. Fazekas de St. Groth, R.G. Webster and A. Datyner, Two new  
staining procedures for quantitative estimation of proteins on  
electrophoretic strips, Biochim. Biophys. Acta 71 (1963), pp. 377-391.


***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***

- Original Message - From: "Gregory Alushin" <[EMAIL PROTECTED] 
>

To: 
Sent: Thursday, September 25, 2008 6:07 PM
Subject: Re: [ccp4bb] Off-topic: coomassie linearity?


The Bio-Rad Flamingo Fluorescent Gel Stain has a very linear  
profile  with protein concentration (if you believe the standard  
curves in the  manual). In my hands it gives nice results for  
binding assays.


Cheers,
-Greg Alushin

On Sep 25, 2008, at 2:35 PM, Jacob Keller wrote:


Dear Crystallographers,

I remember having seen on this listserve that coomassie stain is  
horribly non-linear in intensity per protein concentration, which   
leads me to two questions:


1. Does anybody have a reference for quantitation of coomassie's  
linearity (and possibly other stains), i.e. where and how  
extensive  the linear range is, and

2. Can anybody suggest a more linear protein gel stain?

Thanks,

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***


-
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone:   +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch

[ccp4bb] creating alternate conformations using PyMol

[Neeraj]

Hi all,
 I am working on generating alternate conformations for a 
particular loop region of a protein. I just want to move this region 
into different conformations in order to show the possible states it can 
occur in. Is there an easy way to do this in pymol which will help me 
rotate only a small fragment of the protein while keeping everything 
else static.


thanks for the help in advance!
Neeraj

--
Neeraj Kapoor
TPCB Graduate Fellow
Sakmar Lab/ Molecular Biology & Biochemistry
The Rockefeller University
1230 York Avenue, RRB 510
New York, NY 10021
lab.1.212.327.8284:fax.7904
mobile: 917.535.2030
http://www.rockefeller.edu/labheads/sakmar/sakmar-lab.html