Late induction for short time. Then immediately purify it cut down on any unnecessary steps eg shorter spin all on ice or coldroom etc. Jürgen
...................... 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/ On Feb 15, 2012, at 8:20, "Sivasankar Putta" <sivasankarpu...@iisertvm.ac.in<mailto:sivasankarpu...@iisertvm.ac.in>> wrote: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta <proteingel.pdf>
