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/
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Visiting Professorship in Biophysics, University of South Bohemia (CZ)
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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 I'm
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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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