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Hi Dipankar - I will echo Thuy's suggestion of higher glycerol concentration in your refolding buffer. A protocol I used in a previous lab (for refolding antibody ScFv fragments) steps down gradually over 3 days from 10%-5%-0% glycerol in the dialysis buffer, from an initial sample of inclusion bodies dissolved in 6 M GdnHCl and diluted to 2.4 M GdnHCl with Tris buffer. Your protein may need even higher concentrations. Killikelly A, Zhang H-T, Spurrier B, et al. Thermodynamic Signatures of the Antigen Binding Site of mAb 447–52D Targeting the Third Variable Region of HIV-1 gp120. Biochemistry. 2013;52(36):6249-6257. doi:10.1021/bi400645e. Hope that helps, Cheers, Jared > On Sep 21, 2017, at 6:38 AM, Thuy Ngo <[log in to unmask]> wrote: > > Dear Manna, > > I can see that you use only 5% glycerol in your buffer. This concentration is quite low for non-stable protein. I would suggest you to increase glycerol up to 20%. I had a case before that I need to use 30% glycerol in order to decrease the precipitation. Also, how did you do the refolding? > > Best luck