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
