Hi Meg,
I think with your pi you may also test a Q-Sepharose at neutral to slightly
alkaline pH. Maybe your protein is more comfortable with this condition
compared to a pH of 4.5. As mentioned befor your protein is most likely
precipitated on the column.
Best regards
Christian
Am Dienstag 04 Januar 2011 10:34:26 schrieb megha goyal:
> Dear All,
>
> We used SP sepharose high performance as second stage Ion exchange
> chromatography for polishing the product. We did get pure product but yield
> obtained was mere 25%. Our protein has a pI of 5.5 - 6.0 and we had used 25
> mM Na Acetate buffer pH 4.5 for loading and same buffer with 1M NaCl for
> elution, 25 C.V. linear gradient. Can you suggest some changes that i can
> incorporate to increase the yield i.e additives to be added or some change
> in pH etc. I tried elution with arginine HCL as elution buffer as was
> recommended in one paper, but the yield obtained was even less.
>
> On washing with 2M NaCl ther is not much peak appearing but on washing with
> 1M NaOH substantial peak appears.
>
> Kindly help me through this.
>
>
> meg
>
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