Protein crashing out of solution - (Dec/11/2007 )

Hi all,
I am purifying a protein (MW 32315) on a BioRad NiIDA column and eluting with 250 mM imidazole in 0.5 M NaCl PBS. Within 30 minutes of being eluted, the most concentrated elution fractions (usually about 1 to 3 mg/ml) will crash out of solution.
I usually then spin down those elution fractions and pool with other elution fractions in preparation for dialysis against PBS pH 7.4. The protein then continues to crash out in dialysis.
We have tried altering the pH and the NaCl concentration of the PBS and leaving the protein in 250 mM imidazole but all have the same affect.
Does anyone know of maybe a different buffer that we could dialyse against or give any suggestions as to why the protein crashes so quickly?

Thanks!!!!

Hi

Did u try elution in bigger volume ?

Adding detargents- triton x-100. Tween -20 ?

Did u freeze the elution fraction after elution (might causes aggregates)?

your protein may be misfolded. you may be able to purify it in urea and then dialyze it down to refold.

I like the concept of gradual urea-facilitated refolding while the protein is still on column. Anyone has any luck with that approach?

Hi all,
I am purifying a protein (MW 32315) on a BioRad NiIDA column and eluting with 250 mM imidazole in 0.5 M NaCl PBS. Within 30 minutes of being eluted, the most concentrated elution fractions (usually about 1 to 3 mg/ml) will crash out of solution.
I usually then spin down those elution fractions and pool with other elution fractions in preparation for dialysis against PBS pH 7.4. The protein then continues to crash out in dialysis.
We have tried altering the pH and the NaCl concentration of the PBS and leaving the protein in 250 mM imidazole but all have the same affect.
Does anyone know of maybe a different buffer that we could dialyse against or give any suggestions as to why the protein crashes so quickly?

Thanks!!!!