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 
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* E-mail transmission cannot be guaranteed to be secure or error-free 
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arrive late or incomplete, or contain viruses. The sender therefore 
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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 
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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.
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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 
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individual concerned. The Institute will not accept any liability in respect of 
such communication, and the employee responsible will be personally liable for 
any damages or other liability arising. Employees who receive such an email 
must notify their supervisor immediately.
--

Re: [ccp4bb] 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
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

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



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

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

Re: [ccp4bb] 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] 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