How about incomplete TEV cleavage ? Your protein is a dimer/multimer in solution and some of it is cleaved and some not and they stick together and are separated on the SDS gel ?
Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 27, 2010, at 9:09 AM, vikrant saa wrote: > Dear all > Thanks for yours valuable suggestion. > Just some addition information to make the query clear. > > 1)My protein has 14 cysteine residues. > 2)It is not a metal binding protein. > 3)I have added the protease inhibitor cocktail + PMSF during sonication and > cleavage. I saw only one band of protein bind on beads. Two band appears > after cleavage. > 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in > Rosetta 2DE3 > 5) I have to do MALDI and MS MS to know exact molecular weight and sequence > that differ in both the proteins. Most of the peaks in peptide mass > fingerprint match exactly while some are at different position and size. > Amino acids sequence similarity in both the cases are upto 1-405 amino acids > (as per limitation of peptide mass fingerprint it show some region upto 1-405 > matching in both the proteins). > > > I have certain doubt/solution(s) on the basis of yours feedback and above > information. > > 1) How come PTM can play role when protein expressed in bacteria. > 2) TEV is a very specific protease hence non specific cleavage less likely > occur. > 3) Cysteine residues or temperature dependent expression may be one of the > reason of two band of protein. > > Further suggestion to get rid of this problem will be highly appreciated. > > > With Regards > > Vikrant > > >
