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/
<http://www.uni-luebeck.de/studium/studiengaenge/infection-biology/introduction.html>
Institute of Biochemistry, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany
phone: +49-451-31013105 (secretariate -31013101)
fax: +49-451-31013104
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<Http://www.eine-stadt-sieht-gelb.de>
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Visiting Professorship in Biophysics, University of South Bohemia (CZ)
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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)
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Only two things are infinite, the universe and human stupidity, and
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It is invariably the case that high resolution X-ray structures show
significantly better agreement with solution observables such as
coupling constants, 13C chemical shifts, and proton chemical shifts,
than the corresponding NMR structures, including the very best ones.
Hence, in most cases, a high-resolution crystal structure (< 2.0
Å)will provide a better description of the structure in solution than
the corresponding NMR structure (Kuszewski, Gronenborn & Clore,
1996, Protein Science 5:1067-80)
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Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/
<http://www.uni-luebeck.de/studium/studiengaenge/infection-biology/introduction.html>
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.biochem.uni-luebeck.de>
http://www.eine-stadt-sieht-gelb.de <Http://www.eine-stadt-sieht-gelb.de>
http://www.uni-luebeck.de/studium/studiengaenge/infection-biology
http://www.iobcr.org <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
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is required please request a hard-copy version. Please send us by fax
any message containing deadlines as incoming e-mails are not screened
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* Employees of the Institute are expressly required not to make
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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
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