Hi Megha, how about your protein is stuck to the top of your column ? How does your pressure look like before injection and after injection ? What makes you believe that you have successfully refolded your protein ?
Just some thoughts, Jürgen On May 11, 2010, at 10:53 PM, megha goyal wrote: > Hi all, > > > Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M > cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. > Then we perform concentration using proflux M12 [just concentration and not > diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S > resin [strong cation exchange]. The problem is we do not recover our protein > on performing IEX. HPLC and absorbance reading on concentrate show the > presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and > elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and > even 2M Nacl washing does not show our protein. . Only when we perform NaOH > wash we do see some peak but could not analyse it as it is too alkaline and > cant run on SDS PAGE or HPLC. > > What could be the reason. Where do we lose our protein. Kindly shed some > light on this on where shall I be going wrong. > > thanks and regards, > > meg - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
