[Protein-analysis] Re: Protein Solubility Problem
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Tue Aug 15 07:14:38 EST 2006
rice.jeffrey at gmail.com wrote:
> Elution was in
> 20mM Tris-HCl pH 7.9, 0.5M NaCl, and 1M imidizole. The protein came
> off in Fraction 2, and was (apparently) soluble at that point.
> Analysis by anti-SDF1 WB, coomassie, and ponceau show only the single
> band, so it looks pretty pure.
> I then dialyzed against PBS overnight. I need to do an N-terminal
> coupling with this protein, and the Tris will interfere. During the
> dialysis, the protein came out of solution.
I am not sure why you included the Tris in the first place, imidazole is
a perfectly good buffer.
The second problem is that you changed many things at the same time, so
it is difficult to tell what the cause is. Appart from the changes in
buffer composition it may also be oxydation of thiols in the protein. So
I'd try to dialyse small samples of your protein against buffers where
only one factor was changed, and I'd try DTT (0.1 mM) to protect the
protein. A bit of EDTA or EGTA (say, 1 mM) can also be helpfull as heavy
metal ions can catalyse oxydation.
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