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
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It is invariably the case that high resolution X-ray structures show significantly
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corresponding NMR structure (Kuszewski, Gronenborn & Clore, 1996, Protein Science
5:1067-80)
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