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I have seen this when expressing sub-domains of a larger protein. Adding some (about 0.5% w/v) BOG (beta octylglucoside) worked well for cases similar to what you've described. Cheers, Quyen _______________________________ Quyen Hoang, Ph.D Department of Biochemistry and Molecular Biology, Stark Neurosciences Research Institute Indiana University School of Medicine 635 Barnhill Drive, Room MS0013D Indianapolis, Indiana 46202-5122 Phone: 317-274-4371 Fax: 317-274-4686 email: [log in to unmask] On Jan 8, 2010, at 7:41 AM, Sivaraman Padavattan wrote: > 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 > > >