Properties of proteins purified in imidazole

Achim Recktenwald achimr at home.com
Wed Aug 11 22:42:51 EST 1999

"frank rozsa" <rozsa at UMICH.EDU> wrote in message
news:199908110059.UAA01316 at runningman.rs.itd.umich.edu...
> Hi protein experts,

> 3. I can dialyze the protein out of imidazole (vs 10 mM Tris, 0.5%
>    but when I concentrate using an amicon filter, the sample precipitates.
>    What is a good buffer to store protein samples in at -80?

Your ion concentration is rather low; some proteins like it, others not. I
don't think , anybody has found a rule yet. Try adding some salt (e.g.,
150mM NaCl) or dialyze it agains PBS. You could also try adding up to 20%
glycerol, it often helps keeping proteins in solution. In case your protein
is not properly folded and/or contains free cysteins, you might want to add
a reducing agent, like beta-mercaptoethanol, DTT.

Tris is a very bad buffer for storage at -80C. Due to the dpKa/C value
of -0.031 for Tris , your pH adjusted at 25C will rise by almost 3.3
pH-units at -80C. The dpKa/C for phosphate is only 1/10 of the value for
Tris; therefore, the pH will rise by about 0.33 units.
You can find dpKa/C-values in many buffer listings.

> 4. What about an ice cold acetone precipitation? Is this considered
>    "denaturing"? I've heard that detergents form a slimy, difficult to
>    solubilize pellet in acetone. Any hints about maintaining biological
>    integrity using acetone precipitation?

Acetone precipitation at low temperatures is very mild and not denaturing;
it is in its treatment of proteins similar to ammonium sulfate
precipitation. I would recommend cooling your stock of acetone to -20C, and
if possible your centrifugation  rotor, as well. Otherwise it is very
difficult to keep the temperature sufficiently low during the whole
Why do you need a detergent in your buffer? Are you purifying a membrane
protein? If not, have you tried purifying  it without detergent?



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