Re: [ccp4bb] SP Sep HP tight binding of proteins
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
[ccp4bb] SP Sep HP tight binding of proteins
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
Re: [ccp4bb] SP Sep HP tight binding of proteins
Hi Meg, If the bigger peak is appearing when you wash with 1M NaOH, i think your protein is precipitated on the the column. If you have your protein relatively pure after the precedent step, maybe you can try different buffer. You can probably find a better buffer than this one. If your protein is in good condition before the column, maybe its a problem with the column type. Maybe you can try an other SP. If your protein have Cys, maybe you can try to add som DTT or 2-mercaptoethanol or something like this. In my opinion the best start is to detemined the better pH and after try some other stuff like reducing agent, light detergent... (triton X-100, Glycerol...). Nicolas Le 04/01/11 10:34, megha goyal a écrit : 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 -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
