Likewise, incorporation of 30% (or even more) of ethylene glycol, polyethylene glycol, propylene glycol etc. may save you from proteolysis. This did not work for me but a friend of mine has had very good luck with it. Kind of hit/miss method, though.
Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Christian Biertuempfel Sent: Monday, August 25, 2008 10:22 AM To: [email protected] Subject: Re: [ccp4bb] protein degradation Hi Debajyoti, There is another simple thing you can try: Raise your NaCl concentration to 500 or 1000 mM in your lysis buffer. This helps to clean up your sample further and it might inhibit proteases in your lysate. Good luck, christian Debajyoti Dutta wrote: > > Hi, > > This is going to be an off topic question concerning this community. I > have a protein 6XHis tagged. When retrieved from the Ni-NTA column with > imidazole found to be degraded, appears like a deep band with other > bands (touching each other below the main band) in SDS PAGE. The protein > is a DNA binding (pI~ 10) and Tris-Hcl buffer is used with 10% glycerol > and 300mM NaCl. for purification. Does Phosphate buffer do any help in > stopping the degradation. > > All suggestions are welcome. Thank you for your reply in advance. > > Sincerely > Debajyoti Dutta > > > > Rediff Shopping > <http://adworks.rediff.com/cgi-bin/AdWorks/click.cgi/www.rediff.com/signatur e-home.htm/[EMAIL PROTECTED]/2206641_2199021/2201650/1?PARTNER=3&OAS_QUERY= null> > _______________________________________________________________________ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA _______________________________________________________________________
