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