[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.
[ccp4bb] Postdoctoral position in RNA structural biochemistry at the Karolinska Institutet
A postdoctoral position in RNA structural biochemistry is open in my lab at the Department of Cell and Molecular Biology at the Karolinska Institutet in Stockholm. Successful candidates will participate in all steps involved in structure determination of RNA-protein complexes relevant to tRNA biogenesis. Suitable candidates have a PhD in a relevant area, preferably in RNA biochemistry, structural biology or closely related subjects. A general requirement to be eligible for this position is a doctoral degree obtained after 2008-04-30. The application should contain a cover letter and a CV with publication list. Contact details of two references should also be provided. Send the application as an email with attachments to martin.hallb...@ki.se Review of applications will begin immediately and continue until the position is filled. . B. Martin Hallberg, PhD Department of Cell and Molecular Biology Karolinska Institutet 171 77 Stockholm Sweden http://tinyurl.com/yzn9y5j
