[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

[Hughes, Jon]

yes, "oil of vitriol" is sulphuric acid, "blue vitriol" is copper II sulphate 
as i recall.
j


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von CRAIG A 
BINGMAN
Gesendet: Donnerstag, 30. März 2017 05:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg

Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an 
old name for sulfuric acid.

> On Mar 29, 2017, at 8:40 PM, Keller, Jacob  wrote:
> 
>> And if we are going to pour scorn and vitriol on Tris, why not mention its 
>> large dpKa/dT of 0.03 pH units/deg ?
> 
> Hah! That's what many people are doing when they make buffers: pouring 
> vitriol (HCl) on TRIS! I prefer to pour concentrated HEPES, and get two 
> buffers without adding any extra Cl-.
> 
> JPK

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

[Hughes, Jon]

yes - really, tris should be the buffer of last resort rather than the 
standard. its only general advantages would seem to be that it's cheap and not 
very toxic. 
j

--
Professor Jon Hughes, BSc, PhD
Institute for Plant Physiology
Justus Liebig University, Giessen
Zeughaus, Rm. 341
Senckenbergstr. 3
D35390 Giessen, Germany.
work phone: (+49/0)6419935430
fax:  (+49/0)6419935429
mobile:   (+49/0)1757929098
email:  jon.hug...@uni-giessen.de
homepage:
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Sent without the use of Apple products



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark 
Wilson
Gesendet: Mittwoch, 29. März 2017 22:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg

I heartily concur with Craig.  Tris can be a dangerous buffer for many reasons, 
including those listed below.  In addition, as a primary amine, it can 
complicate work with metalloproteins and has moderate nucleophilicity.  There 
is almost always a better buffer choice than Tris.
Best regards,
Mark

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN"
 wrote:

>
>
>
>There are almost always better choices than Tris buffer.
>
>
>Mo Cleland used to call it “Trash” buffer.  He is no longer with us, 
>but today I will happily carry that flag in his honor.
>
>
>Tris may show up in your crystal structure, especially at carbohydrate 
>binding sites.
>Tris may be a surprisingly strong competitive inhibitor in your enzyme 
>assays, especially as above.
>Tris has an absolutely miserably bad change in pKa vs. temperature.  It 
>is larger than -0.03 pKa/dT(C).  It can be a catastrophically bad 
>choice for flash-freezing protein aliquots.
>
>
>If you taken the time and incurred the expense of preparing a 
>macromolecular sample for crystallization studies, and you are worried 
>about the price difference between Tris and HEPES, in my opinion you 
>are absolutely worried about the wrong things.
>
>
>Why are people substantially concerned about the buffering capacity of 
>a buffer for final sample preparation?  You have a purified protein, 
>presumably without substrate present.  Exactly what do you think is 
>generating or absorbing hydrogen ions  in that solution?  Oxidation of 
>reducing agent should be about the only thing that is taxing the 
>buffer.  From the example below, oxidation of 5 mM BME will put some 
>pressure on the buffer, but unfortunately Tris accelerates the 
>oxidation of BME relative,  to, say, HEPES. And surely you aren’t just 
>letting the protein sit and oxidize in the refrigerator? Oh you might 
>be since when you tried to snap freeze it in Tris, it turned into 
>cooked egg white because the pH went to over 10 before it vitrified.
>(http://www.sciencedirect.com/science/article/pii/S0031942200801429)
>
>
>Isn’t the whole point to use a small amount of buffer so you can easily 
>push the pH around in crystallization screens? (At which point the 
>sample is usually in 100+ mM buffer.)
>
>
>On Mar 29, 2017, at 2:03 PM, Hughes, Jon 
> wrote:
>
>...it's just a wonderful tradition! there's an interesting description 
>of the history of tris in maniatis
>cheers
>jon
> 
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im  Auftrag von 
>David Briggs
>Gesendet: Mittwoch, 29. März 2017 17:53
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: Re: [ccp4bb] protein precipitation reg
> 
>It doesn't cost as much as HEPES, iirc.
>On Wed, 29 Mar 2017, 16:36 Keller, Jacob, 
>wrote:
>
>
>A bit off topic, but I’ve always wondered how TRIS got so popular what 
>with it’s pKa of 8.3—does anyone know?
> 
>JPK
> 
>From: CCP4
> bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger  
>Rowlett
>Sent: Wednesday, March 29, 2017 11:10 AM
>To: CCP4BB@JISCMAIL.AC.UK
>Subject: Re: [ccp4bb] protein precipitation reg
>
>
>
>
> 
>What are you dialyzing against? Your storage solution should typically 
>be buffered away from the pI and contain at least a small amount of 
>kosmotropic salt, e.g. NaCl. Some proteins will require additional 
>stabilizing/solubilizing  agents such as glycerol or reducing agents. 
>FYI, Tris-Cl, pH 7.5 has very little buffer capacity (about 15% of the 
>total concentration in the acid direction). We typically use Tris-Cl pH 
>8.0, which is closer to the Tris pKa and has good buffer capacity for  
>both acid and base. For pH 7.5 we would typically use HEPES as the 
>storage buffer.
>
>___
>Roger S. Rowlett
>Gordon & Dorothy Kline Professor
>Department of Chemistry
>Colgate University
>13 Oak Drive
>Hamilton, NY 13346
>
>tel: (315)-228-7245
>ofc: (315)-228-7395
>fax: (315)-228-7935
>email: rrowl...@colgate.edu
>On 3/

[ccp4bb] PSI Career Return program

[Dmitry Veprintsev]

Dear All,
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best regards, Dmitry
-- 
Dr. Dmitry Veprintsev
Group Leader
Conformational Dynamics of GPCRs
Laboratory of Biomolecular Research, OFLC/103
Paul Scherrer Institut
5232 Villigen PSI
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dmitry.veprint...@psi.ch
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​​
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Re: [ccp4bb] [ccp4bb] protein precipitation reg

[Keller, Jacob]

Darn it, you're right.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Thursday, March 30, 2017 4:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

yes, "oil of vitriol" is sulphuric acid, "blue vitriol" is copper II sulphate 
as i recall.
j


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von CRAIG A 
BINGMAN
Gesendet: Donnerstag, 30. März 2017 05:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg

Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an 
old name for sulfuric acid.

> On Mar 29, 2017, at 8:40 PM, Keller, Jacob  wrote:
> 
>> And if we are going to pour scorn and vitriol on Tris, why not mention its 
>> large dpKa/dT of 0.03 pH units/deg ?
> 
> Hah! That's what many people are doing when they make buffers: pouring 
> vitriol (HCl) on TRIS! I prefer to pour concentrated HEPES, and get two 
> buffers without adding any extra Cl-.
> 
> JPK

Re: [ccp4bb] protein precipitation reg

[Antonio Ariza]

I personally like TRIS for the first few steps of purification and then change 
to something else during my last dialysis step. I mostly work with bacteria and 
they often produce lysates that have pH's that are too acidic for good nickel 
affinity chromatography, which is why I use 100 mM TRIS pH > 7.8 at this point 
(if I remember correctly, histidines won't be properly charged and won't bind 
well to the nickel ions at pH < 7.6). 10 or 50 mM of most buffers might not 
actually buffer a fairly concentrated bacterial lysate and therefore produce a 
solution that is more acidic than expected.



I also run size exclusion chromatography in 100 mM TRIS to reduce the cost as 
1L of 100 mM HEPES is fairly expensive (pH can be lowered at this point 
depending on the protein's PI, but I like to have the solution strongly 
buffered). After SEC I will use 10 mM HEPES (or other appropriate buffers) for 
the final dialysis step. This reduces the cost and works well for me.



BTW, TRIS is obviously not a good buffer choice for ion exchange chromatography 
(which relies on precise pH differences between two buffers) because the pH of 
a TRIS solution will change with even small fluctuations in temperature.



Best regards,



Tony



--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of 
DNA



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chun Luo 
[c...@accelagen.com]
Sent: 29 March 2017 22:15
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg

In addition to price, the prevalence of Ni purification may be another reason 
for Tris popularity. Some His-tagged constructs don't bind to Ni well in HEPES. 
I wonder if anyone has similar experience or comments. --Chun

Re: [ccp4bb] Large number of outliers in the dataset

[Xiao Lei]

This case is encouraging to me that a structure can be solved with such
high mosaicity (in your report is 1.9). I wonder how the diffraction looks
like (I imagine spots smearing or streak). With such high mosaicity, the
unit cell dimension and space group determination is highly likely not
accurate. I usually lose hope of a dataset with average mosaicity above 1.3
and I would not process any high mosaic data, but from your case it seems
2.6A cutoff you get a very nice solution.  It seems the  data processing
software (Imosflm or XDS) nowadays can handle with high mosaic data well.





On Wed, Mar 29, 2017 at 11:00 AM, Gianluca Santoni  wrote:

> In addition to what Nicolas has pointed out, is it quite suspicious to me
> that you have the same multiplicity in the high resolution shell as in the
> low resolution.
>
> On Mar 29, 2017 18:17, Nicolas FOOS  wrote:
>
> Dear Juliana,
>
> all the statistics presented here looks good in terms of resolution cut
> (maybe I will be less sever). For me the point is about the mosaicity you
> report 1.90 it's high in my opinion. How looks you images? I am wondering
> if the indexation is really right. And maybe the complain of Xtriage about
> outlier is due to this high mosaicity. What is the diagnostic of Xtriage in
> terms of possible twinning? I am also wondering about a pseudo translation.
> Maybe try to re-processed your data in this direction.
>
> Hope to help.
>
> Nicolas
>
> Nicolas Foos
> PhD
> Structural Biology Group
> European Synchrotron Radiation Facility (E.S.R.F)
> 71, avenue des Martyrs
> CS 40220
> 38043 GRENOBLE Cedex 9+33 (0)6 76 88 14 87 <+33%206%2076%2088%2014%2087>+33 
> (0)4 76 88 45 19 <+33%204%2076%2088%2045%2019>
>
> On 29/03/2017 17:56, Mark J van Raaij wrote:
>
> To be really convinced I think you should also compare the maps at 2.6 and
> 2.3 Å. If the 2.3 Å map looks better, go for it. If it doesn’t look better,
> perhaps you are adding noise, but the I/sigma and CC1/2 values suggest you
> aren’t.
> Perhaps try 2.5 and 2.4 Å also.
> And perhaps remove a well-ordered aa from the input model, refine at
> different resolutions and compare the difference maps for that aa. Or
> calculate omit maps at different resolutions and compare those.
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
>
> On 29 Mar 2017, at 17:44, Phil Evans  wrote:
>
> It is not clear to me why you believe that cutting the resolution of the
> data would improve your model (which after all is the aim of refinement).
> At the edge CC(1/2) and I/sigI are perfectly respectable, and there doesn’t
> seem to be anything wrong with the Wilson plot. Th R-factor will of course
> be higher if you include more weak data, but minimising R is _not_ the aim
> of refinement. You should keep all the data
>
> I don’t know what xtriage means by “large number of outliers”: perhaps
> someone else can explain
>
> Phil
>
>
>
> On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira <
> juliana.olive...@lnbio.cnpem.br> wrote:
>
> Hello,
> I have one dataset at 2.3 Å (probably it can be better, I/σ = 2.1 and
> CC1/2 = 0.779, the summary data is below), but when I perform Xtriage
> analysis it says that “There are a large number of outliers in the data”.
> The space group is P212121. When I refine the MR solution the Rfree stops
> around 30% and it doesn´t decrease (in fact if I continue refining it
> starts to increase).
> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>
> 
>
> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify
> about outliers anymore. I could refine the MR solution very well, the final
> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a
> good structure.
> I run Zanuda to confirm the space group and it says that the space group
> assignment seems to be correct.
> Do you think that I can improve my structure and solve it at 2.3 Å or
> better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to
> justify the resolution cut, right? What should I say?
> Thank you for your help!
> Regards,
> Juliana
>
> Summary data:
> OverallInnerShell  OuterShell
> Low resolution limit  51.51  51.51
>   2.42
> High resolution limit  2.30 7.27
>2.30
> Rmerge   0.147
>   0.054   0.487
> Rmerge in top intensity bin0.080   -
>  -
> Rmeas (within I+/I-)  0.155   0.057
>   0.516
> Rmeas (all I+ & I-)0.155   0.057
>   0.516
> Rpim (within I+/I-)0.048   0.017
>   0.164

Re: [ccp4bb] protein precipitation reg

[Antonio Ariza]

If I remember correctly, Triton X-100 (or any other surfactant for that matter) 
is a bad idea for protein intended for crystallography. I can't remember the 
paper, but I'm sure I read that somebody showed it's basically impossible to 
remove all of the surfactant molecules from the protein no matter how you 
dialyse it. These large floppy molecules stick to your protein molecules and 
interfere with methods such as mass spec and can hinder the crystallisation 
process.



I'm not sure this is your case, but it might help. If your protein has a very 
high (or low) PI, it will probably need a strongly ionic environment to be 
stable. I work with nucleic acid binding proteins and in general I find they do 
better in buffers with concentrations > 500 mM NaCl until all contaminating 
proteins have been removed. Only after SEC do I lower the NaCl concentration to 
between 50 and 150 mM NaCl (you'll need to test which final concentration works 
best for your protein).



In any case, some precipitation is normal and you can easily remove it by 
centrifugation. An ultracentrifuge works best, but even a benchtop centrifuge 
for eppendorfs can remove most of the precipitate from your dialysed solution.



Best Regards,



Tony



--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of 
DNA

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Akilandeswari 
Gopalan [akilaibt2...@gmail.com]
Sent: 30 March 2017 07:02
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg

Dear all,
I have used the following buffers for purification and dialysis. this is fyi.

Lysis buffer:
25mM Tris pH 7 or 7.5 or 8
100-500mM NaCl (increase in salt concentration increased precipitation of the 
protein in the column itself)
 5mM Beta mercaptoethanol
0.5% Triton X 100
I have tried with other buffers also.

a. HEPES buffer pH7.5

b. Phosphate buffer pH 7.8

c. MOPS buffer pH 8

Wash and Elution Buffer:
25mM Tris pH 7 or 7.5 or 8
100-500mM NaCl
20 and 30mM Imidazole for wash
300mM for elution


Dialysis Buffer:

1. Tris 25mM pH 7

2. Tris 25mM pH 7.5

3. Tris 25mM pH 8

4. Tris 25mM pH 7.5, 5% glycerol

5. Tris 25mM pH 7.5, 10% glycerol

6. Tris 25mM pH 7.5, 20% glycerol

7. Tris 25mM pH7.5, 50mM NaCl

8. Tris 25mM pH7.5, 100mM NaCl

9. Tris 25mM pH7.5, 1mM MgCl2

10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM L-Glu


In all these cases the protein precipitates. i have tried to do buffer exchange 
also. i can see precipitate sticking on the walls of the tube during the 
process.

Re: [ccp4bb] protein precipitation reg

[mesters]

If the pI of the protein is below the pH of the buffer (net negatively 
charged protein), optimum stabilization (salting out; lower solubility) 
of the macromolecule is achieved by combining a kosmotropic anion with a 
chaotropic cation, e.g. Ammoniumsulfate (most successful salt)!


/For your pI 9.7 protein: Vice versa/, if the pI of the protein is above 
the pH of the buffer (net positively charged protein and thus inversion 
of the Hofmeister series), 50-150 mM Ammoniumsulfate is a far better 
choice for solubilisation than NaCl.//


/For your pI 5.6 protein:/Maybe you need a stronger "solubilizer" salt 
such as Nitrate or Thiocyanate while increasing the pH to 8.0 or 8.5 (to 
increase the net charge of the protein).


Good luck,

Jeroen


Am 29.03.17 um 15:38 schrieb Akilandeswari Gopalan:

Dear all,
I am a PhD student doing structural studies on a few proteins from 
Mycobacterium tuberculosis. The gene encoding the proteins I work on 
are cloned into pet22b with c terminal His tag. the proteins are 
expressing well. upon purification I am getting good yield of protein 
but during dialysis, the proteins precipitate. Kindly suggest some 
solutions to avoid aggregation. pI of one protein is 9.7 and that of 
the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer 
with 20-30mM imidazole for washing and 300mM imidazole for eluting the 
proteins.


Thank you
Regards
Akila

--
Akilandeswari G




--
Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/ 



Institute of Biochemistry, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany
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It is invariably the case that high resolution X-ray structures show 
significantly better agreement with solution observables such as 
coupling constants, 13C chemical shifts, and proton chemical shifts, 
than the corresponding NMR structures, including the very best ones. 
Hence, in most cases, a high-resolution crystal structure (< 2.0 Å)will 
provide a better description of the structure in solution than the 
corresponding NMR structure  (Kuszewski, Gronenborn & Clore, 1996, 
Protein Science 5:1067-80)

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Re: [ccp4bb] protein precipitation reg

[Kittikhun Wangkanont]

Hi Akila,
I'm curious about your choice of pET22b. If you cut out the signal peptide
that comes with the vector and express your protein in the cytoplasm, then
what I am about to say doesn't apply. However, if you are exporting the
protein into the periplasm, have you considered doing osmotic shock instead
of lysing the cells? When you lyse the cells, you are likely to get
misfolded/unprocessed protein from the cytoplasm that will likely
precipitate.
Pun

On Wed, Mar 29, 2017 at 8:38 PM, Akilandeswari Gopalan <
akilaibt2...@gmail.com> wrote:

> Dear all,
> I am a PhD student doing structural studies on a few proteins from
> Mycobacterium tuberculosis. The gene encoding the proteins I work on are
> cloned into pet22b with c terminal His tag. the proteins are expressing
> well. upon purification I am getting good yield of protein but during
> dialysis, the proteins precipitate. Kindly suggest some solutions to avoid
> aggregation. pI of one protein is 9.7 and that of the other is 5.6
> I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM
> beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with
> 20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.
>
> Thank you
> Regards
> Akila
>
> --
> Akilandeswari G
>
>

[ccp4bb] ligand with out space group information

[chemocev marker]

Hi
I have model of ligand molecule and it does not open in coot. Its not a
crystal structure. I can view it in the pymol or chimera but not in the
coot. It gives error that it does not have any space group information. Is
there is a way to open it in coot.

best

Jiri

Re: [ccp4bb] protein precipitation reg

[Debanu]

Yes, I once spent quite a bit of time engineering mutations into my target to 
improve solubility to exclude detergent from the purification to improve 
crystal growth and diffraction: 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374227/

If you think buffer conditions hold the key to your solubility woes over other 
factors, you can look into Hampton's (or other vendors) Solubility and 
Stability Kit:
https://hamptonresearch.com/documents/product/hr008095_binder1.pdf

Or the pH Slice Kit:
https://hamptonresearch.com/product_detail.aspx?cid=30&sid=200&pid=616

Best,
Debanu


> On Mar 30, 2017, at 4:47 AM, Antonio Ariza  
> wrote:
> 
> If I remember correctly, Triton X-100 (or any other surfactant for that 
> matter) is a bad idea for protein intended for crystallography. I can't 
> remember the paper, but I'm sure I read that somebody showed it's basically 
> impossible to remove all of the surfactant molecules from the protein no 
> matter how you dialyse it. These large floppy molecules stick to your protein 
> molecules and interfere with methods such as mass spec and can hinder the 
> crystallisation process.
>  
> I'm not sure this is your case, but it might help. If your protein has a very 
> high (or low) PI, it will probably need a strongly ionic environment to be 
> stable. I work with nucleic acid binding proteins and in general I find they 
> do better in buffers with concentrations > 500 mM NaCl until all 
> contaminating proteins have been removed. Only after SEC do I lower the NaCl 
> concentration to between 50 and 150 mM NaCl (you'll need to test which final 
> concentration works best for your protein).
>  
> In any case, some precipitation is normal and you can easily remove it by 
> centrifugation. An ultracentrifuge works best, but even a benchtop centrifuge 
> for eppendorfs can remove most of the precipitate from your dialysed solution.
>  
> Best Regards,
>  
> Tony
>  
> --
> 
> Dr. Antonio Ariza
> University of Oxford
> Sir William Dunn School of Pathology
> South Parks Road
> Oxford
> OX1 3RE
> e-mail: antonio.ar...@path.ox.ac.uk
> Tel: 00 +44 1865 285655
>  
> Links to my public profiles:
> ResearchGate
> LinkedIn
> GoogleScholar
> Twitter
>  
> Check out my latest paper!!!
> The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Akilandeswari 
> Gopalan [akilaibt2...@gmail.com]
> Sent: 30 March 2017 07:02
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] protein precipitation reg
> 
> Dear all,
> I have used the following buffers for purification and dialysis. this is fyi.
> 
> Lysis buffer:
> 25mM Tris pH 7 or 7.5 or 8
> 100-500mM NaCl (increase in salt concentration increased precipitation of the 
> protein in the column itself)
>  5mM Beta mercaptoethanol 
> 0.5% Triton X 100 
> I have tried with other buffers also.
> a. HEPES buffer pH7.5
> b. Phosphate buffer pH 7.8
> c. MOPS buffer pH 8
>  
> Wash and Elution Buffer:
> 25mM Tris pH 7 or 7.5 or 8
> 100-500mM NaCl 
> 20 and 30mM Imidazole for wash
> 300mM for elution
>  
>  
> Dialysis Buffer:
> 1. Tris 25mM pH 7
> 2. Tris 25mM pH 7.5
> 3. Tris 25mM pH 8
> 4. Tris 25mM pH 7.5, 5% glycerol
> 5. Tris 25mM pH 7.5, 10% glycerol
> 6. Tris 25mM pH 7.5, 20% glycerol
> 7. Tris 25mM pH7.5, 50mM NaCl
> 8. Tris 25mM pH7.5, 100mM NaCl
> 9. Tris 25mM pH7.5, 1mM MgCl2
> 10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM L-Glu
> 
> In all these cases the protein precipitates. i have tried to do buffer 
> exchange also. i can see precipitate sticking on the walls of the tube during 
> the process. 

Re: [ccp4bb] ligand with out space group information

[Paul Emsley]

On 30/03/17 14:59, chemocev marker wrote:

Hi


Hi.

I have model of ligand molecule and it does not open in coot. Its not 
a crystal structure. I can view it in the pymol or chimera but not in 
the coot. It gives error that it does not have any space group 
information. Is there is a way to open it in coot.




If there is not a CRYST1 card in your pdb file, then Coot will warn you 
that your molecule does not have symmetry.  It is merely a warning, not 
an error.


If coot can't read the pdb file it's likely (it seems to me) that it's 
not actually a (specifications-compliant) pdb file.  Coot should give 
you an error about any line that it particularly doesn't like.



Paul.

[ccp4bb] binding protein to nickel column

[Shubhangi Agarwal]

Hello

I have a 10kda histidine kinase domain protein with a pI of 9.5.
It has a C-term his tag and despite using different buffers the protein
doesnt bind to the nickel cloumn. it comes out in the flow trhough.
Buffers used- 50mM tris Ph=8, 300mM NaCl
   50mM tris Ph=7, 300mM NaCl
50mM  hepes Ph=7.5, 300mM NaCl
50mM tris Ph=8, 300mM NaCl, 10% glycerol
Can someone suggest to get ensure binding of the protein to the nickel-nta
column

Shubhangi
PhD student
under Dr. Jhimli Dasgupta
St. Xavier's College
Kolkata
India

Re: [ccp4bb] binding protein to nickel column

[Edward Pryor]

Hi, Shubhangi,

One possibility is that the his-tag could be non-accessible.  Have you tried 
moving to the tag to the other end of the protein (N vs C)?  Another thing that 
worked for me for a past project is using a small amount (1-2M) of urea for the 
IMAC step and then dialyzing away the urea before proceeding onto the other 
purification steps.  This increased protein binding to the IMAC column and 
after dialyzing away the urea, the protein still retained full activity (I was 
also able to crystallize and determine the structure of it).

Hope this helps.
--Eddie


Edward E. Pryor, Ph.D.
Anatrace
Field Applications Scientist
Cell: 434.270.2511
Find Anatrace on LinkedIn



From: CCP4 bulletin board  on behalf of Shubhangi 
Agarwal 
Reply-To: Shubhangi Agarwal 
Date: Thursday, March 30, 2017 at 3:20 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] binding protein to nickel column

Hello

I have a 10kda histidine kinase domain protein with a pI of 9.5.
It has a C-term his tag and despite using different buffers the protein doesnt 
bind to the nickel cloumn. it comes out in the flow trhough.
Buffers used- 50mM tris Ph=8, 300mM NaCl
   50mM tris Ph=7, 300mM NaCl
50mM  hepes Ph=7.5, 300mM NaCl
50mM tris Ph=8, 300mM NaCl, 10% glycerol
Can someone suggest to get ensure binding of the protein to the nickel-nta 
column

Shubhangi
PhD student
under Dr. Jhimli Dasgupta
St. Xavier's College
Kolkata
India

Re: [ccp4bb] binding protein to nickel column

[PULSARSTRIAN]

Hi Shubhangi,

 As Edward suggested, you can try with N-terminal
His tag. For this you can either clone in N-terminal His tagged based
vectors or by site directed insertion of 18 nucleotides coding for 6 His at
the N-terminus region.


But, before that, I suggest you to try with *CAPS buffer pH
11.0*. The change in net charge on your protein might improve binding to Ni
column.

Good luck.


Regards,

Bhanu

On Fri, Mar 31, 2017 at 12:50 AM, Shubhangi Agarwal <
shubhangiagarwal2...@gmail.com> wrote:

> Hello
>
> I have a 10kda histidine kinase domain protein with a pI of 9.5.
> It has a C-term his tag and despite using different buffers the protein
> doesnt bind to the nickel cloumn. it comes out in the flow trhough.
> Buffers used- 50mM tris Ph=8, 300mM NaCl
>50mM tris Ph=7, 300mM NaCl
> 50mM  hepes Ph=7.5, 300mM NaCl
> 50mM tris Ph=8, 300mM NaCl, 10% glycerol
> Can someone suggest to get ensure binding of the protein to the nickel-nta
> column
>
> Shubhangi
> PhD student
> under Dr. Jhimli Dasgupta
> St. Xavier's College
> Kolkata
> India
>



-- 
B4U

Re: [ccp4bb] protein precipitation reg

[Edward A. Berry]

On 03/30/2017 08:10 AM, mesters wrote:

If the pI of the protein is below the pH of the buffer (net negatively charged 
protein), optimum stabilization (salting out; lower solubility) of the 
macromolecule is achieved by combining a kosmotropic anion with a chaotropic 
cation, e.g. Ammoniumsulfate (most successful salt)!


??
According to the wikipedia page on Hoffmeister series, NH4+ is one of the 
_least_ chaotropic cations.


/For your pI 9.7 protein: Vice versa/, if the pI of the protein is above the pH 
of the buffer (net positively charged protein and thus inversion of the 
Hofmeister series), 50-150 mM Ammoniumsulfate is a far better choice for 
solubilisation than NaCl.//



That would explain why it is so hard to precipitate cytochrome c with NH4SO4!


/For your pI 5.6 protein:/Maybe you need a stronger "solubilizer" salt such as 
Nitrate or Thiocyanate while increasing the pH to 8.0 or 8.5 (to increase the net charge 
of the protein).

Good luck,

Jeroen


Am 29.03.17 um 15:38 schrieb Akilandeswari Gopalan:

Dear all,
I am a PhD student doing structural studies on a few proteins from 
Mycobacterium tuberculosis. The gene encoding the proteins I work on are cloned 
into pet22b with c terminal His tag. the proteins are expressing well. upon 
purification I am getting good yield of protein but during dialysis, the 
proteins precipitate. Kindly suggest some solutions to avoid aggregation. pI of 
one protein is 9.7 and that of the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with 
20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.

Thank you
Regards
Akila

--
Akilandeswari G




--
Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/ 


Institute of Biochemistry, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany
phone: +49-451-31013105 (secretariate -31013101)
fax: +49-451-31013104

http://jobs.zeit.de/image-upload/logo_10564.jpg
http://www.biochem.uni-luebeck.de 
http://www.eine-stadt-sieht-gelb.de 
http://www.uni-luebeck.de/studium/studiengaenge/infection-biology
http://www.iobcr.org 

Visiting Professorship in Biophysics, University of South Bohemia (CZ)
--
If you can look into the seeds of time and tell which grain will grow and which 
will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act 
I, Scene 3)
--
Only two things are infinite, the universe and human stupidity, and I'm not 
sure about the former (Albert Einstein)
--
It is invariably the case that high resolution X-ray structures show significantly 
better agreement with solution observables such as coupling constants, 13C chemical 
shifts, and proton chemical shifts, than the corresponding NMR structures, including 
the very best ones. Hence, in most cases, a high-resolution crystal structure (< 
2.0 Å)will provide a better description of the structure in solution than the 
corresponding NMR structure  (Kuszewski, Gronenborn & Clore, 1996, Protein Science 
5:1067-80)
--
Disclaimer
* This message contains confidential information and is intended only for the 
individual named. If you are not the named addressee you should not 
disseminate, distribute or copy this e-mail. Please notify the sender 
immediately by e-mail if you have received this e-mail by mistake and delete 
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individual concerned. The Institute will not accept any liability in respect of 
such communication, and the employee responsible will be personally liable for 
any damages or other liability arising. Employees who receive such an email 
must notify their supervisor immediately.
--

Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card under Windows 10

[Xiao Lei]

Hi All,

I summarized my Coot and Pymol 3D experience here. Monitor is from Asus 24
or 27 inch 3D monitor. Monitor works with Nvidia 3D kit. Refresh rate set
120 HZ. Please first download and install Nvidia 3D kit driver and Asus
monitor driver and install first when working with Windows 7 or 10 (you can
find drivers from official website).  Even after install drivers, 3D is not
automatically working in Windows, you need to follow
http://www.ysbl.york.ac.uk/~lohkamp/coot/wincoot-faq.html#mozTocId906434
and do some settings.

1. Windows 7 with Quadro M4000, Display port (DP) on PC to DP on Monitor,
Works very stable with Coot and Pymol. No 3-D mini DIN cable needed.

2. Window 7 with Quadro 5000, DVI-D dual link on PC to DVI-D dual link on
Monitor. Works very stable with Coot and Pymol. No 3-D mini DIN cable
needed.

3. Fedora 24 with Quadro 5000, DVI-D dual link on PC to DVI-D dual link on
Monitor. Works very stable with Coot and Pymol. 3-D mini DIN cable is
needed in this case. Do not dnf update kernel, as most of the time you have
to re-install Nvidia driver again after kernel updated.

4. Ubuntu Mate with Quadro 5000, DVI-D dual link on PC to DVI-D dual link
on Monitor. Works very stable with Coot and Pymol. 3-D mini DIN cable is
needed in this case. In Ubuntu Mate, the installation of Nvidia driver can
be automatically fetch by the system (install proprietary driver), no need
to type command as in Fedora.

5. Windows 10 with Quadro M4000, Display port (DP) on PC to DP on Monitor,
Works with Pymol 3D but Not Coot in my case. No 3-D mini DIN cable needed.
Not stable even with Pymol 3D, I need to reset every parameter after update
and reset refresh rate to 120hz.. This is the worst option in my case!
Because Win10 keeps giving us problem I switch back to Win7 and everything
works great!




On Thu, Mar 2, 2017 at 4:15 PM, Xiao Lei  wrote:

> Hi All,
>
> Just share my experience, Windows 10 works for pymol 3D under Quadro M4000
> with Nvidia 3D kit (no need to connect the 3 pin MINI DIN, Quadro M4000
> does not have 3 pin Mini DIN connections). I use Asus 24 inch 3D monitor,
> connect the monitor with display port to display port of Quadro M4000, no
> active DP to DVI needed in my case. We have a problem for Coot 3D works
> even when Pymol 3D works ok, we are trying to figure out the problem but
> Windows 10 does work for pymol 3D under DP to DP port.
>
> On Tue, Jan 31, 2017 at 6:39 AM, Yong Wang  wrote:
>
>> Xiao,
>>
>>
>>
>> I had a direct connection from display port to display port that had
>> worked for several years (Windows 7).  Since last year I have been losing
>> the stereo.  Often the control panel won’t show the 120 Hz like what you
>> saw.  Sometimes I was able to forcefully add a 120 Hz resolution (using the
>> “Customize …” menu).  But it was not stable, i.e. the stereo would stay for
>> some time then disappear and go back to the state of not having 120 Hz.  I
>> have not had time to investigate further into this but I suspect a driver
>> issue here.  You may want to check the drivers for both the graphics card
>> and the monitor.
>>
>>
>>
>> Yong
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Xiao Lei
>> *Sent:* Monday, January 30, 2017 11:50 PM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>>
>> *Subject:* Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card
>> under Windows 10
>>
>>
>>
>> Taka,
>>
>>
>>
>> I really appreciate this information! I do have Displayport cable and can
>> connect the card and Asus VG248QE both via the DP cable, somehow on the
>> Nvidia control panel in Windows 10, I do not have an option of choosing
>> "120HZ" or "144Hz", I only have option choosing "60Hz" or below, which
>> sounds weird to me. Windows 10 should make things easier not harder. If
>> CentOS 6 works then Windows 10 should work, I guess I have to go back to
>> Windows 7 if needed, but as I already ordered the DP to DVI active cable,
>> I'll test this cable first and update CCP4bb later.
>>
>>
>>
>> On Mon, Jan 30, 2017 at 7:50 PM,  wrote:
>>
>> Xiao,
>>
>>
>>
>> If you connect the board and monitor by DisplayPort cable directly, it
>> should work.
>>
>> I confirmed with Quadro M4000 and BenQ XL2420Z on CentOS 6, though not
>> tested on Windows.
>>
>>
>>
>> Taka
>>
>>
>>
>> *From:* Xiao Lei [mailto:xiaolei...@gmail.com]
>> *Sent:* Tuesday, January 31, 2017 10:08 AM
>> *Cc:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* Re: [ccp4bb] Coot and Pymol 3D in Quadro M4000 Graphics Card
>> under Windows 10
>>
>>
>>
>> I changed my mind, I should order the usb- powered DP to DVI dual link...
>>
>>
>>
>> On Mon, Jan 30, 2017 at 5:02 PM, Christine Gee  wrote:
>>
>> Hi Xiao,
>>
>>
>>
>> I can confirm you need to buy an active adapter if you want to convert
>> the display port on the graphics card to DVI. The one that they supply with
>> the graphics card is passive and won't work. I was in the same boat about a
>> year ago. I bought a USB powered active adapter which allowed 120htz. My

[ccp4bb] Protein Ligand interaction using SPR technique

[Praveen Tripathi]

Dear all,
Sorry for off-topic question.
I want to study protein interaction with few small molecules using SPR
(Machine- *BIACORE 3000*).

The recombinant protein expressed in bacterial expression system is of 92
kDa (with His tag), pI= 9.

*Question-*
1. What should be the chip of choice- *NTA chip or CM5 chip* of GE
healthcare?
2. Is there any alternative available for chips and machine?

Thanks in advance.

Regards

Praveen

Re: [ccp4bb] Protein Ligand interaction using SPR technique

[David Briggs]

Hi Praveen,

The BiaCore 3000 is an older model, and is not optimised for protein/small
molecule interactions. The BiaCore T-100 and T-200 have enhanced
sensitivity and are better suited small molecules (assuming you have to use
SPR).

As for chip choice, it depends an awful lot on the behaviour of your
protein. NTA imo is not great as you will always have some loss if protein
during the experiment - covalent immobilisation to CM5 is preferable.

Do you have access to ITC? You might consider this if protein is easy to
make. It's a better experiment than SPR, but is much more thirsty.

HTH,

Dave

On Fri, 31 Mar 2017, 06:04 Praveen Tripathi, 
wrote:

> Dear all,
> Sorry for off-topic question.
> I want to study protein interaction with few small molecules using SPR
> (Machine- *BIACORE 3000*).
>
> The recombinant protein expressed in bacterial expression system is of 92
> kDa (with His tag), pI= 9.
>
> *Question-*
> 1. What should be the chip of choice- *NTA chip or CM5 chip* of GE
> healthcare?
> 2. Is there any alternative available for chips and machine?
>
> Thanks in advance.
>
> Regards
> 
> Praveen
>
-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs

Re: [ccp4bb] protein precipitation reg

[mesters]

Please have a look at the very elaborated/detailed discussion by Martin 
Chaplin on chaotropes and kosmotropes


http://www1.lsbu.ac.uk/water/kosmotropes_chaotropes.html

and on the Hofmeister series

http://www1.lsbu.ac.uk/water/hofmeister_series.html

and *note the diference* in the way the ions are ranked.

Happy reading,

Jeroen




Am 30.03.17 um 23:36 schrieb Edward A. Berry:



On 03/30/2017 08:10 AM, mesters wrote:
If the pI of the protein is below the pH of the buffer (net 
negatively charged protein), optimum stabilization (salting out; 
lower solubility) of the macromolecule is achieved by combining a 
kosmotropic anion with a chaotropic cation, e.g. Ammoniumsulfate 
(most successful salt)!



??
According to the wikipedia page on Hoffmeister series, NH4+ is one of 
the _least_ chaotropic cations.


/For your pI 9.7 protein: Vice versa/, if the pI of the protein is 
above the pH of the buffer (net positively charged protein and thus 
inversion of the Hofmeister series), 50-150 mM Ammoniumsulfate is a 
far better choice for solubilisation than NaCl.//




That would explain why it is so hard to precipitate cytochrome c with 
NH4SO4!


/For your pI 5.6 protein:/Maybe you need a stronger "solubilizer" 
salt such as Nitrate or Thiocyanate while increasing the pH to 8.0 or 
8.5 (to increase the net charge of the protein).


Good luck,

Jeroen


Am 29.03.17 um 15:38 schrieb Akilandeswari Gopalan:

Dear all,
I am a PhD student doing structural studies on a few proteins from 
Mycobacterium tuberculosis. The gene encoding the proteins I work on 
are cloned into pet22b with c terminal His tag. the proteins are 
expressing well. upon purification I am getting good yield of 
protein but during dialysis, the proteins precipitate. Kindly 
suggest some solutions to avoid aggregation. pI of one protein is 
9.7 and that of the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same 
buffer with 20-30mM imidazole for washing and 300mM imidazole for 
eluting the proteins.


Thank you
Regards
Akila

--
Akilandeswari G




--
Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/ 



Institute of Biochemistry, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany
phone: +49-451-31013105 (secretariate -31013101)
fax: +49-451-31013104

http://jobs.zeit.de/image-upload/logo_10564.jpg
http://www.biochem.uni-luebeck.de 
http://www.eine-stadt-sieht-gelb.de 


http://www.uni-luebeck.de/studium/studiengaenge/infection-biology
http://www.iobcr.org 

Visiting Professorship in Biophysics, University of South Bohemia (CZ)
--
If you can look into the seeds of time and tell which grain will grow 
and which will not, speak then to me who neither beg nor fear 
(Shakespeare's Macbeth, Act I, Scene 3)

--
Only two things are infinite, the universe and human stupidity, and 
I'm not sure about the former (Albert Einstein)

--
It is invariably the case that high resolution X-ray structures show 
significantly better agreement with solution observables such as 
coupling constants, 13C chemical shifts, and proton chemical shifts, 
than the corresponding NMR structures, including the very best ones. 
Hence, in most cases, a high-resolution crystal structure (< 2.0 
Å)will provide a better description of the structure in solution than 
the corresponding NMR structure  (Kuszewski, Gronenborn & Clore, 
1996, Protein Science 5:1067-80)

--
Disclaimer
* This message contains confidential information and is intended only 
for the individual named. If you are not the named addressee you 
should not disseminate, distribute or copy this e-mail. Please notify 
the sender immediately by e-mail if you have received this e-mail by 
mistake and delete this e-mail from your system.
* E-mail transmission cannot be guaranteed to be secure or error-free 
as information could be intercepted, corrupted, lost, destroyed, 
arrive late or incomplete, or contain viruses. The sender therefore 
does not accept liability for any errors or omissions in the contents 
of this message, which arise as a result of e-mail transmission. If 
verification is required please request a hard-copy version. Please 
send us by fax any message containing deadlines as incoming e-mails 
are not screened for response deadlines.
* Employees of the Institute are expressly required not to make 
defamatory statements and not to infringe or authorize any 
infringement of copyright or any other legal right by email 
communications. Any such communication is contrary to Institute 
policy and outside the scope of the employment of the individual 
concerned. The Institute will not accept any liability in respect of 
such communication, and the employee responsible will be personally 
liable for any damages or other liability arising. Employees who 
receive su

Re: [ccp4bb] Protein Ligand interaction using SPR technique

[M T]

Dear Praveen,

As said, Biacore3000 is not the best for small molecules, but in some case you 
can do nice things with this system. I see 3 possibilities :
- You can immobilize your molecules on the chip if you have an amine on it 
which is supposed to not participate to the interaction.
- You can have also the possibility to use carrier protein as BSA which is 
often found labeled with various small molecules and why not yours.
- You can also use competition assays if your small molecules are supposed to 
inhibit larger molecules interaction.

I am agree with the problems of non stable baseline with NiNTA chips, covalent 
interactions are better, but in these cases regeneration can be more 
difficult...

An other thing you can consider is streptavidin surfaces, especially if you can 
obtain your small molecules biotinylated. But in that case, long linkers should 
be used in case of your small molecules may be buried in an hydrophobic pocket.

If you cannot fit in these case you should consider other experiments as ITC, 
NMR (STD transfert)...

Good luck.

Michel.

> Le 31 mars 2017 à 07:01, Praveen Tripathi  a 
> écrit :
> 
> Dear all,
> Sorry for off-topic question.
> I want to study protein interaction with few small molecules using SPR 
> (Machine- BIACORE 3000).
> 
> The recombinant protein expressed in bacterial expression system is of 92 kDa 
> (with His tag), pI= 9.
> 
> Question- 
> 1. What should be the chip of choice- NTA chip or CM5 chip of GE healthcare?
> 2. Is there any alternative available for chips and machine?
> 
> Thanks in advance.
> 
> Regards
> 
> Praveen