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 

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 
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 such an email 
must notify their supervisor immediately.
--

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=200=616

Best,
Debanu


> On Mar 30, 2017, at 4:47 AM, Antonio Ariza <antonio.ar...@path.ox.ac.uk> 
> 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] 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
>
>

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
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 such an email must notify their 
supervisor immediately.

--

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<mailto:antonio.ar...@path.ox.ac.uk>
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza>
LinkedIn<https://www.linkedin.com/in/antonioariza1>
GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en>
Twitter<https://twitter.com/DrAntonioAriza?lang=en>

Check out my latest paper!!!
The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of 
DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5>

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

[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<mailto:antonio.ar...@path.ox.ac.uk>
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza>
LinkedIn<https://www.linkedin.com/in/antonioariza1>
GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0J=en>
Twitter<https://twitter.com/DrAntonioAriza?lang=en>

Check out my latest paper!!!
The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of 
DNA<http://www.cell.com/molecular-cell/abstract/S1097-2765(16)30722-5>



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

[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:
http://www.uni-giessen.de/fbz/fb08/Inst/pflphys/pflaphygroups/ag-hughes
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"
<CCP4BB@JISCMAIL.AC.UK on behalf of cabing...@wisc.edu> 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 
><jon.hug...@bot3.bio.uni-giessen.de> 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, <kell...@janelia.hhmi.org>
>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 t

[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 <kell...@janelia.hhmi.org> 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

[Paul Miller]

Only one of your dialysis buffers has a decent (100 mM) NaCl. Maybe you could 
try higher salt IN COMBINATION with glycerol. There's also NDSBs that stabilise 
proteins.

Also, could the high immidazole be keeping the protein happy? You could test 
this by dialysing into the same buffer with a range of immidazole 
concentrations. You could try other related natural compounds, histidine, 
histamine, as well to understand this process better.

Cheers, Paul

Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Thu, 30 Mar 2017 11:32:05 +0530
>From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Akilandeswari 
>Gopalan <akilaibt2...@gmail.com>)
>Subject: Re: [ccp4bb] protein precipitation reg  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   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 MgCl[2
>
>   ]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. 
>
>   On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das
>   <debanu@gmail.com> wrote:
>
> Hi Akila,
>
> In addition to what others have asked about the
> dialysis buffer, a few
> more comments that might help to decide next steps
> because the
> precipitation (note precipitation and aggregation
> are related but not
> synonymous) may be due to several different or
> related reasons:
>
> 1) At what stage are you dialyzing? Is it after
> SEC? Could your
> protein be too concentrated at that point since
> your yield is high
> leading to some precipitation? How severe is the
> loss?
> 2) Did you try more purification before dialysis?
> 3) Are you removing the detergent in the buffer
> you are dialyzing against?
> 4) Can you try buffer exchange during
> concentration instead of dialysis?
> 5) Try increasing your NaCl concentration and
> adding 5-10% glycerol to
> improve protein solubility.
> 6) Did you try cleaving the C-term His-tag before
> dialysis? Did you
> try N-term His tag?
> 7) Do you really need to dialyze? Did you try
> assays or
> crystallization trials with the purification
> buffer? You can run a SEC
> column without imidazole to remove that before
> crystallization.
> 8) PSI/SBKB TargetTrack can be great resource to
> look at
> expression/purification protocols for similar
> proteins:
> http://sbkb.org/tt/
> 9) Also look up the Tb Structural Genomics
> resource to see if there is
> anything on this target or related targets:
> http://www.webtb.org/
>
> Best,
> Debanu
>
> On Wed, Mar 29, 2017 at 6:38 AM, 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
> >
>
>   --
>   Akilandeswari G
>   JRF
>   C/O Dr. Alamelu Raja
>   National Institute for Research in Tuberculosis
>   Chetput, Chennai

Re: [ccp4bb] protein precipitation reg

[Akilandeswari Gopalan]

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.

On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das  wrote:

> Hi Akila,
>
> In addition to what others have asked about the dialysis buffer, a few
> more comments that might help to decide next steps because the
> precipitation (note precipitation and aggregation are related but not
> synonymous) may be due to several different or related reasons:
>
> 1) At what stage are you dialyzing? Is it after SEC? Could your
> protein be too concentrated at that point since your yield is high
> leading to some precipitation? How severe is the loss?
> 2) Did you try more purification before dialysis?
> 3) Are you removing the detergent in the buffer you are dialyzing against?
> 4) Can you try buffer exchange during concentration instead of dialysis?
> 5) Try increasing your NaCl concentration and adding 5-10% glycerol to
> improve protein solubility.
> 6) Did you try cleaving the C-term His-tag before dialysis? Did you
> try N-term His tag?
> 7) Do you really need to dialyze? Did you try assays or
> crystallization trials with the purification buffer? You can run a SEC
> column without imidazole to remove that before crystallization.
> 8) PSI/SBKB TargetTrack can be great resource to look at
> expression/purification protocols for similar proteins:
> http://sbkb.org/tt/
> 9) Also look up the Tb Structural Genomics resource to see if there is
> anything on this target or related targets: http://www.webtb.org/
>
> Best,
> Debanu
>
> On Wed, Mar 29, 2017 at 6:38 AM, Akilandeswari Gopalan
>  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
> >
>



-- 
Akilandeswari G
JRF
C/O Dr. Alamelu Raja
National Institute for Research in Tuberculosis
Chetput, Chennai

Re: [ccp4bb] protein precipitation reg

[Debanu Das]

Hi Akila,

In addition to what others have asked about the dialysis buffer, a few
more comments that might help to decide next steps because the
precipitation (note precipitation and aggregation are related but not
synonymous) may be due to several different or related reasons:

1) At what stage are you dialyzing? Is it after SEC? Could your
protein be too concentrated at that point since your yield is high
leading to some precipitation? How severe is the loss?
2) Did you try more purification before dialysis?
3) Are you removing the detergent in the buffer you are dialyzing against?
4) Can you try buffer exchange during concentration instead of dialysis?
5) Try increasing your NaCl concentration and adding 5-10% glycerol to
improve protein solubility.
6) Did you try cleaving the C-term His-tag before dialysis? Did you
try N-term His tag?
7) Do you really need to dialyze? Did you try assays or
crystallization trials with the purification buffer? You can run a SEC
column without imidazole to remove that before crystallization.
8) PSI/SBKB TargetTrack can be great resource to look at
expression/purification protocols for similar proteins:
http://sbkb.org/tt/
9) Also look up the Tb Structural Genomics resource to see if there is
anything on this target or related targets: http://www.webtb.org/

Best,
Debanu

On Wed, Mar 29, 2017 at 6:38 AM, Akilandeswari Gopalan
 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
>

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[CRAIG A BINGMAN]

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] [ccp4bb] protein precipitation reg

[Keller, Jacob]

>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] [ccp4bb] protein precipitation reg

[Janet Newman]

Just to point out that whatever buffer you purify your protein into is possibly 
not the one that will keep your protein happiest.  We had the opportunity of 
testing about 250 proteins in DSF against 26 different buffer / salt 
combinations (in triplicate, with lots of controls) and found out that about 
1/3 of the time the protein was significantly (4C or more) stable in some other 
buffer system than the one it was in.
Ristic, M., Rosa, N., Seabrook, S.A., Newman, J., 2015. Formulation screening 
by differential scanning fluorimetry: how often does it work? Acta 
Crystallographica Section F Structural Biology Communications 71, 1359–1364. 
doi:10.1107/S2053230X15012662

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 ?

Cheers, Janet

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CRAIG A 
BINGMAN
Sent: Thursday, 30 March 2017 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg


> On Mar 29, 2017, at 4:15 PM, Chun Luo <c...@accelagen.com> wrote:
> 
> 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

No, I have not specifically noted that before.

Additionally, why would you use a positively charged buffer on a weak cation 
exchange resin?  The Ni affinity resins, in addition to their noteworthy 
affinity for various metals, are also weak cation exchange resins.  Binding a 
positively charged buffer to a negatively charged column material can cause pH 
effects that you probably weren’t expecting.

Tris may have some uses.  But using it in a mobile phase with Ni affinity 
columns or as a final sample buffer aren’t the best cases for choosing Tris.  I 
understand it is widely used in aquaculture applications where they are 
treating tens of thousands of gallons of water, and price of the buffer 
substance is an actual consideration.

Craig

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[CRAIG A BINGMAN]

> On Mar 29, 2017, at 4:15 PM, Chun Luo  wrote:
> 
> 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

No, I have not specifically noted that before.

Additionally, why would you use a positively charged buffer on a weak cation 
exchange resin?  The Ni affinity resins, in addition to their noteworthy 
affinity for various metals, are also weak cation exchange resins.  Binding a 
positively charged buffer to a negatively charged column material can cause pH 
effects that you probably weren’t expecting.

Tris may have some uses.  But using it in a mobile phase with Ni affinity 
columns or as a final sample buffer aren’t the best cases for choosing Tris.  I 
understand it is widely used in aquaculture applications where they are 
treating tens of thousands of gallons of water, and price of the buffer 
substance is an actual consideration.

Craig

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[Chun Luo]

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

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark 
Wilson
Sent: Wednesday, March 29, 2017 1:12 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: 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"
<CCP4BB@JISCMAIL.AC.UK on behalf of cabing...@wisc.edu> 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 
><jon.hug...@bot3.bio.uni-giessen.de> 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, <kell...@janelia.hhmi.org>
>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

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[Mark Wilson]

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"
<CCP4BB@JISCMAIL.AC.UK on behalf of cabing...@wisc.edu> 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
><jon.hug...@bot3.bio.uni-giessen.de> 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, <kell...@janelia.hhmi.org>
>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 3/29/2017 9:38 AM, Akilandeswari Gopalan 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
>pre

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[CRAIG A BINGMAN]

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 
<jon.hug...@bot3.bio.uni-giessen.de<mailto:jon.hug...@bot3.bio.uni-giessen.de>> 
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<mailto: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, 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Roger 
Rowlett
Sent: Wednesday, March 29, 2017 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto:rrowl...@colgate.edu>
On 3/29/2017 9:38 AM, Akilandeswari Gopalan 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

--


Fehler! Es wurde kein Dateiname angegeben.




David Briggs PhD
Fehler! Es wurde kein Dateiname 
angegeben.<http://angegeben.about.me/david_briggs>about.me/david_briggs<http://angegeben.about.me/david_briggs>

[ccp4bb] AW: [ccp4bb] protein precipitation reg

[Hughes, Jon]

...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, 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Roger 
Rowlett
Sent: Wednesday, March 29, 2017 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto: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<mailto:rrowl...@colgate.edu>
On 3/29/2017 9:38 AM, Akilandeswari Gopalan 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

--


Fehler! Es wurde kein Dateiname angegeben.
[Das Bild wurde vom Absender entfernt.]



David Briggs PhD
Fehler! Es wurde kein Dateiname angegeben.about.me/david_briggs

Re: [ccp4bb] protein precipitation reg

[Roger Rowlett]

Exactly. Tris is very cheap. HEPES not so much. On the other hand, 
zwitterionic buffers have significant advantages in terms of controlling 
inorganic anion or cation concentrations.


___
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 3/29/2017 11:53 AM, David Briggs wrote:

It doesn't cost as much as HEPES, iirc.

On Wed, 29 Mar 2017, 16:36 Keller, Jacob, <kell...@janelia.hhmi.org 
<mailto:kell...@janelia.hhmi.org>> 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
<mailto:CCP4BB@JISCMAIL.AC.UK>] *On Behalf Of *Roger Rowlett
*Sent:* Wednesday, March 29, 2017 11:10 AM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto: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 <mailto:rrowl...@colgate.edu>

On 3/29/2017 9:38 AM, Akilandeswari Gopalan 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

--
--

David Briggs PhD
https://about.me/david_briggs

<https://about.me/david_briggs?promo=email_sig_source=email_sig_medium=email_sig_campaign=external_links>

Re: [ccp4bb] protein precipitation reg

[David Briggs]

It doesn't cost as much as HEPES, iirc.

On Wed, 29 Mar 2017, 16:36 Keller, Jacob, <kell...@janelia.hhmi.org> 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 3/29/2017 9:38 AM, Akilandeswari Gopalan 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
>
>
>
-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs
<https://about.me/david_briggs?promo=email_sig_source=email_sig_medium=email_sig_campaign=external_links>

Re: [ccp4bb] protein precipitation reg

[Bonsor, Daniel]

Probably the price, HEPES is nearly 5-8 fold more expensive. PBS is fine, 
unless you have to add divalents. TRIS is your (cheap) friend!

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Wednesday, March 29, 2017 11:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein precipitation reg

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<mailto: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<mailto:rrowl...@colgate.edu>
On 3/29/2017 9:38 AM, Akilandeswari Gopalan 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

Re: [ccp4bb] protein precipitation reg

[Keller, Jacob]

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<mailto:rrowl...@colgate.edu>
On 3/29/2017 9:38 AM, Akilandeswari Gopalan 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

Re: [ccp4bb] protein precipitation reg

[Roger Rowlett]

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 3/29/2017 9:38 AM, Akilandeswari Gopalan 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

Re: [ccp4bb] protein precipitation reg

[Keller, Jacob]

And what are you dialyzing it against?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Akilandeswari Gopalan
Sent: Wednesday, March 29, 2017 9:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein precipitation reg

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] protein precipitation reg

[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

Re: [ccp4bb] Protein precipitation

[Manjula Ramu]

Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein
sample and found more amount in soluble form itself, however within a day
protein gets degraded. If I have to further purify the protein I have to
concentrate the IMAC elutes and concentrate using filters. In this step
protein degradation was more and filter used to block and could not
complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN bhanu.hydpri...@gmail.com
wrote:

 Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to
 1mM for IMAC using Ni-NTA. If your protein requires any additional
 co-factors (like Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

 On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu manjula@gmail.com
 wrote:

 Hi all,

 I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm
 NaCl, 5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed
 affinity purification. Eluted with 200mM imidazole.  While elution I could
 see slight turbid in eluted protein (I get pure protein, single band on
 SDS-PAGE). However, when I centrifuged I couldn't see any pellet. After
 some time(a day) protein got degraded. In other method I tried cation
 exchange purification of lysate with MES buffer pH 6.5. Here also I saw
 slight precipitation and later degradation.

 After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0,
 here also same problem I faced.

 Please suggest me a method where I can get stable protein.

 Thanks and Regards,
 Manjula R
 Research Scholar
 Department of Biophysics
 National Institute of Mental Health and Neurosciences
 Bengaluru-29, Karnataka
 INDIA
 E-mail: manjula@gmail.com
 Mobile no:+91-9538553356
 http://www.nimhans.kar.nic.in/
 https://www.nimhans.kar.nic.in/




 --
 B4U

[ccp4bb] Protein precipitation

[Michel, Max]

Hi Manjula,

I had a similar problem: pH, salt, additives and protease inhibitors (I tried 
them all) didn't work. The protein degraded after lysis of bacteria within 20 
min at 24°C to 50 %. I had luck after i cooled down all buffers and materials 
to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h 
in icewaterbath). I performed IMAC, IEX and SEC to get rid of the degradation. 
Finally the Protein was stable for at least one month.
You have to take care about temperature induced pH changes. Some buffers change 
their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can lead to 
precipitation if you are working close to the pI of your protein.

Maybe you can try to characterize the protease to find a suitable protease 
inhibitor. Make some massspectrometry with your degraded sample. An other 
option is to test via westernblot. If you have some C- oder N-terminal 
monoclonal well characterized antibody.

Greets Max

Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] im Auftrag von Manjula Ramu 
[manjula@gmail.com]
Gesendet: Dienstag, 19. Mai 2015 08:05
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Protein precipitation

Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein 
sample and found more amount in soluble form itself, however within a day 
protein gets degraded. If I have to further purify the protein I have to 
concentrate the IMAC elutes and concentrate using filters. In this step protein 
degradation was more and filter used to block and could not complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN 
bhanu.hydpri...@gmail.commailto:bhanu.hydpri...@gmail.com wrote:
Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
for IMAC using Ni-NTA. If your protein requires any additional co-factors (like 
Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:
Hi all,

I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 
5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity 
purification. Eluted with 200mM imidazole.  While elution I could see slight 
turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.

After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here 
also same problem I faced.

Please suggest me a method where I can get stable protein.

Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/



--
B4U





Forschungszentrum Juelich GmbH
52425 Juelich
Sitz der Gesellschaft: Juelich
Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498
Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher
Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender),
Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt,
Prof. Dr. Sebastian M. Schmidt

Re: [ccp4bb] Protein precipitation

[S. Mohanty]

Hi Manjula,
I will lyse the bacterials cells and monitor the degradation of the lysed 
protein in lysis buffer over 4-5 days before proceeding with purification.  
What is the lifetime of this expressed protein once lysed?  How long can it 
stay after lysis without degradation or aggregation? 
 Does this protein contain disulfide bonds?  How many?  How big is this protein?
Smita Smita Mohanty, Ph.D.  Associate Professor Department of Chemistry447 
Physical SciencesOklahoma State UniversityStillwater, OK 74078-3071 Phone: 
(405) 744 6636E-mail: Smita.Mohanty@okstate.eduWeb: 
chemistry.okstate.edu/mohanty                            


 On Monday, May 18, 2015 11:49 PM, Manjula Ramu manjula@gmail.com 
wrote:
   

 Hi all,I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 
mm NaCl, 5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed 
affinity purification. Eluted with 200mM imidazole.  While elution I could see 
slight turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 
6.0, here also same problem I faced.Please suggest me a method where I can get 
stable protein.
Thanks and Regards,Manjula R 
Research Scholar
Department of Biophysics 
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula.iob@gmail.comMobile no:+91-9538553356
http://www.nimhans.kar.nic.in/


  

Re: [ccp4bb] Protein precipitation

[Reza Khayat]

Hi,

Try using 100mM Ammonium Citrate pH 8.5 as the buffer for your lysis -add salts 
as well. The Citrate will inhibit metalloproteases and is Ni-NTA friendly. It 
solved our proteolysis problems and may help with yours.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
85 St. Nicholas Dr. CDI 12308
New York, NY 10031
(212) 650-6070
www.khayatlab.orghttp://www.khayatlab.org

On May 19, 2015, at 2:05 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:

Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein 
sample and found more amount in soluble form itself, however within a day 
protein gets degraded. If I have to further purify the protein I have to 
concentrate the IMAC elutes and concentrate using filters. In this step protein 
degradation was more and filter used to block and could not complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN 
bhanu.hydpri...@gmail.commailto:bhanu.hydpri...@gmail.com wrote:
Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
for IMAC using Ni-NTA. If your protein requires any additional co-factors (like 
Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:
Hi all,

I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 
5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity 
purification. Eluted with 200mM imidazole.  While elution I could see slight 
turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.

After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here 
also same problem I faced.

Please suggest me a method where I can get stable protein.

Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/



--
B4U

Re: [ccp4bb] Protein precipitation

[Fischmann, Thierry]

The information about of the cleavage site can be used in two ways : possibly 
identify the protease - as it has already been suggested - or introduce a 
mutation at the cleavage site which would prevent clipping from occurring.

The caveat of introducing a mutation is that there is always the possibility 
that the mutated residue plays a key role.

Good luck
Thierry

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. 
Rice
Sent: Tuesday, May 19, 2015 12:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein precipitation

For one of our more easily-degraded proteins, we added a mix of protease 
inhibitors (those expensive tablets you can buy) and also put a drop of EDTA 
into each tube in the fraction collector so that any contaminating 
metal-dependent proteases would be knocked out as soon as the protein came off 
the metal affinity column.  That seemed to help.
Also, if you can get your protein to stick to the next column in whatever 
buffer it comes off the IMAC column in, there's no need to dialyze or 
concentration between columns - the faster you get it clean, the better.
Good luck!
 Phoebe Rice


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel, Max 
[m.mic...@fz-juelich.de]
Sent: Tuesday, May 19, 2015 1:32 AM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein precipitation
Hi Manjula,

I had a similar problem: pH, salt, additives and protease inhibitors (I tried 
them all) didn't work. The protein degraded after lysis of bacteria within 20 
min at 24°C to 50 %. I had luck after i cooled down all buffers and materials 
to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h 
in icewaterbath). I performed IMAC, IEX and SEC to get rid of the degradation. 
Finally the Protein was stable for at least one month.
You have to take care about temperature induced pH changes. Some buffers change 
their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can lead to 
precipitation if you are working close to the pI of your protein.

Maybe you can try to characterize the protease to find a suitable protease 
inhibitor. Make some massspectrometry with your degraded sample. An other 
option is to test via westernblot. If you have some C- oder N-terminal 
monoclonal well characterized antibody.

Greets Max

Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] im Auftrag von Manjula Ramu 
[manjula@gmail.com]
Gesendet: Dienstag, 19. Mai 2015 08:05
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Protein precipitation
Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein 
sample and found more amount in soluble form itself, however within a day 
protein gets degraded. If I have to further purify the protein I have to 
concentrate the IMAC elutes and concentrate using filters. In this step protein 
degradation was more and filter used to block and could not complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN 
bhanu.hydpri...@gmail.commailto:bhanu.hydpri...@gmail.com wrote:
Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
for IMAC using Ni-NTA. If your protein requires any additional co-factors (like 
Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:
Hi all,

I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 
5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity 
purification. Eluted with 200mM imidazole.  While elution I could see slight 
turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.

After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here 
also same problem I faced.

Please suggest me a method where I can get stable protein.

Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula

Re: [ccp4bb] Protein precipitation

[Phoebe A. Rice]

For one of our more easily-degraded proteins, we added a mix of protease 
inhibitors (those expensive tablets you can buy) and also put a drop of EDTA 
into each tube in the fraction collector so that any contaminating 
metal-dependent proteases would be knocked out as soon as the protein came off 
the metal affinity column.  That seemed to help.
Also, if you can get your protein to stick to the next column in whatever 
buffer it comes off the IMAC column in, there's no need to dialyze or 
concentration between columns - the faster you get it clean, the better.
Good luck!
 Phoebe Rice


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel, Max 
[m.mic...@fz-juelich.de]
Sent: Tuesday, May 19, 2015 1:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein precipitation

Hi Manjula,

I had a similar problem: pH, salt, additives and protease inhibitors (I tried 
them all) didn't work. The protein degraded after lysis of bacteria within 20 
min at 24°C to 50 %. I had luck after i cooled down all buffers and materials 
to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h 
in icewaterbath). I performed IMAC, IEX and SEC to get rid of the degradation. 
Finally the Protein was stable for at least one month.
You have to take care about temperature induced pH changes. Some buffers change 
their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can lead to 
precipitation if you are working close to the pI of your protein.

Maybe you can try to characterize the protease to find a suitable protease 
inhibitor. Make some massspectrometry with your degraded sample. An other 
option is to test via westernblot. If you have some C- oder N-terminal 
monoclonal well characterized antibody.

Greets Max

Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] im Auftrag von Manjula Ramu 
[manjula@gmail.com]
Gesendet: Dienstag, 19. Mai 2015 08:05
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Protein precipitation

Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein 
sample and found more amount in soluble form itself, however within a day 
protein gets degraded. If I have to further purify the protein I have to 
concentrate the IMAC elutes and concentrate using filters. In this step protein 
degradation was more and filter used to block and could not complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN 
bhanu.hydpri...@gmail.commailto:bhanu.hydpri...@gmail.com wrote:
Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
for IMAC using Ni-NTA. If your protein requires any additional co-factors (like 
Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:
Hi all,

I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 
5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity 
purification. Eluted with 200mM imidazole.  While elution I could see slight 
turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.

After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here 
also same problem I faced.

Please suggest me a method where I can get stable protein.

Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/



--
B4U





Forschungszentrum Juelich GmbH
52425 Juelich
Sitz der Gesellschaft: Juelich
Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498
Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher
Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender),
Karsten Beneke (stellv

Re: [ccp4bb] Protein precipitation

[Mark J van Raaij]

a third way to use the information would be to reclone the part before and/or 
after the protease cleavage site, i.e. you may have identified (a) stable 
domain(s) that may crystallize much better than the full-length protein.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij








On 19 May 2015, at 18:48, Fischmann, Thierry wrote:

 The information about of the cleavage site can be used in two ways : possibly 
 identify the protease - as it has already been suggested - or introduce a 
 mutation at the cleavage site which would prevent clipping from occurring.
  
 The caveat of introducing a mutation is that there is always the possibility 
 that the mutated residue plays a key role.
  
 Good luck
 Thierry
  
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe 
 A. Rice
 Sent: Tuesday, May 19, 2015 12:23 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Protein precipitation
  
 For one of our more easily-degraded proteins, we added a mix of protease 
 inhibitors (those expensive tablets you can buy) and also put a drop of EDTA 
 into each tube in the fraction collector so that any contaminating 
 metal-dependent proteases would be knocked out as soon as the protein came 
 off the metal affinity column.  That seemed to help.  
 Also, if you can get your protein to stick to the next column in whatever 
 buffer it comes off the IMAC column in, there's no need to dialyze or 
 concentration between columns - the faster you get it clean, the better.
 Good luck!
  Phoebe Rice
  
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel, Max 
 [m.mic...@fz-juelich.de]
 Sent: Tuesday, May 19, 2015 1:32 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Protein precipitation
 
 Hi Manjula,
 
 I had a similar problem: pH, salt, additives and protease inhibitors (I tried 
 them all) didn't work. The protein degraded after lysis of bacteria within 20 
 min at 24°C to 50 %. I had luck after i cooled down all buffers and materials 
 to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h 
 in icewaterbath). I performed IMAC, IEX and SEC to get rid of the 
 degradation. Finally the Protein was stable for at least one month. 
 You have to take care about temperature induced pH changes. Some buffers 
 change their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can 
 lead to precipitation if you are working close to the pI of your protein.
 
 Maybe you can try to characterize the protease to find a suitable protease 
 inhibitor. Make some massspectrometry with your degraded sample. An other 
 option is to test via westernblot. If you have some C- oder N-terminal 
 monoclonal well characterized antibody. 
 
 Greets Max
 Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] im Auftrag von Manjula 
 Ramu [manjula@gmail.com]
 Gesendet: Dienstag, 19. Mai 2015 08:05
 An: CCP4BB@JISCMAIL.AC.UK
 Betreff: Re: [ccp4bb] Protein precipitation
 
 Thanks all for the suggestions.
 @ Pius,
 I used bacterial expression system.Yes I centrifuged precipitated protein 
 sample and found more amount in soluble form itself, however within a day 
 protein gets degraded. If I have to further purify the protein I have to 
 concentrate the IMAC elutes and concentrate using filters. In this step 
 protein degradation was more and filter used to block and could not complete 
 the process
 .
 Do you use batch method of elution or gradient???
 Also do you add PMSF in the elution buffer???
  
 Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
 tried using 500mM NaCl too but no change was observed.
  
 
 Thanks and Regards,
 Manjula R 
 Research Scholar
 Department of Biophysics 
 National Institute of Mental Health and Neurosciences
 Bengaluru-29, Karnataka
 INDIA
 E-mail: manjula@gmail.com
 Mobile no:+91-9538553356
 http://www.nimhans.kar.nic.in/
  
 On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN bhanu.hydpri...@gmail.com 
 wrote:
 Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
 for IMAC using Ni-NTA. If your protein requires any additional co-factors 
 (like Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.
  
 On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu manjula@gmail.com wrote:
 Hi all,
 I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm 
 NaCl, 5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed 
 affinity purification. Eluted with 200mM imidazole.  While elution I could 
 see slight turbid in eluted protein (I get pure protein, single band on 
 SDS-PAGE). However, when I centrifuged I couldn't see any pellet. After some 
 time(a day) protein got degraded. In other method I tried cation exchange 
 purification of lysate with MES buffer pH 6.5. Here also I saw slight 
 precipitation and later degradation.
 After this again I