I assumed, perhaps wrongly, that the fusion was homogeneous prior to cleavage as the existence of the MBP band post-cleavage would not have been noteworthy if it had also been there pre-cleavage.
A time course or at least a time zero sample is always a good idea. Truncation products prior to proteolysis can certainly complicate interpretation of results. In my experience, precipitation of the target protein upon proteolysis from an MBP fusion is a fairly common problem and not necessarily fixable. Cynthia Sent from my iPhone On Nov 4, 2012, at 1:19 PM, "Bosch, Juergen" <jubo...@jhsph.edu<mailto:jubo...@jhsph.edu>> wrote: @Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the MBP band also just before TEV addition - it might also be truncation products which we happen to see all the time. The ratio varies depending on the construct but it can be as bad as a 1:1 ratio. You can really only tell if TEV cleaves if you do a time course experiment at RT with a defined amount of your protein and see if the fusion construct decreases. An alternative for the lack of your 17kDa desired band is simply your fusion construct is cleaved but your cleaved product might a) not be soluble at that pH or b) aggregates and precipitates. You might be able to perform the cleavage on the Amylose column keeping a constant flow cycling. Jürgen ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu