Hey Dipankar, There are various additives for improving solubility. I would consider some of these:
- denaturants (ie urea, guanidine) - aggregation suppressors (arginine, low concentrations of urea, etc) - folding enhancers (sucrose, ammonium sulfate) And have you tried refolding your protein on the column, during purification? This has the advantage of allowing you to transition into any number of buffer systems as you go from the unfolded to folded state, without having protein-protein interactions that may otherwise cause precipitation. Corbin Black, BSc MSc Biochemistry Candidate University of Northern British Columbia Department of Chemistry bla...@unbc.ca<mailto:bla...@unbc.ca> blackcor...@gmail.com<mailto:blackcor...@gmail.com> 250-552-5046 Sent from my iPhone On Sep 21, 2017, at 3:14 AM, Dipankar Manna <dipankar.biot...@gmail.com<mailto:dipankar.biot...@gmail.com>> wrote: Hi, I am working with a serine protease. As the protein is not soluble I am purifying it from the inclusion bodies followed by refolding. The protein shows good activity after refolding but the major concern is the 'precipitation'. Though the protein express quite well, but I loose almost 50-60% protein during refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room temperature. I tried refolding at 4 degree, but it even end up with more precipitant. It also precipitates during concentrating, so in general I almost loose most of the protein during this refolding and concentration steps. I start with 6 lit culture that give around 1-2 mg protein in the final step, which I am not happy with. ? Any suggestion to deal this issue would be highly appreciated. Thank you in advance. Best, Dipankar? -- Dipankar Manna, Ph.D Postdoctoral Researcher Department of Molecular Medicine Institute of Basic Medical Sciences University of Oslo, Domus Medica Oslo, Norway Mob : +47 451 66 517<tel:451%2066%20517> E-mail: dipankar.ma...@medisin.uio.no<mailto:dipankar.ma...@kjemi.uio.no> dipankar.biot...@gmail.com<mailto:dipankar.biot...@gmail.com> http://www.med.uio.no/imb/english/people/aca/dipankam/
