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 >
