you may try partial denaturation during purification steps ( i.e. to include 3 M urea in your gel filtration or ion exchange or N-nta buffers), that may loosen up the interactions without complete denaturation. You may afterward dialyse to remove urea.
r Ravindra D. Makde Currently, Postdoctoral fellow, Tan Lab, Dept of Biochem & Mol biology, The Pennsylvania State University, University Park, PA, US. Tel: 814-777-1776 (mobile) email: [email protected] Scientific Officer E, High Pressure Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085 INDIA Tel: +91 22 25593761 (Lab) email: [email protected] [email protected] --- On Fri, 1/8/10, Sivaraman Padavattan <[email protected]> wrote: > From: Sivaraman Padavattan <[email protected]> > Subject: [ccp4bb] Reg. Protein purification > To: [email protected] > Date: Friday, January 8, 2010, 7:41 AM > Dear all, > I am trying to express the human protein using bacterial > expression strain (Rosetta) and purified using Ni-NTA > affinity purification. The Molecular weight of out protein > is 47 kDa. In SDS-PAGE, we have seen that 27 kDa contaminant > protein co-eluted with our protein even at high > concentration of Imidazole. In Superose 12 column, these two > proteins eluted as a single peak and its corresponding > molecular weight suggestive of partial interaction. By mass > mapping we have found 27 kDa band is an adenylate kinase. Is > there any specific way to separate adenylate kinase from > our protein? > > Thanks in advance, > > Sivaraman Padavattan > > > > >
