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Do you see the same MBP band (or corresponding to the same MW, in case it isn't MBP) before cleavage, at the same total concentration of protein? If not, your protein could be crashing out. Even so, if you use SDS and heat up the sample, I am surprised that your protein just disappeared. TEV protease has a pretty long recognition sequence. It doesn't appear as is in your protein, does it? Is it possible to attach the tag at the other terminus? Perhaps it is more accessible? Of course, it still doesn't solve the mystery of the disappearing protein if the band is indeed MBP that appears after the cleavage reaction. On Sun, Nov 4, 2012 at 3:07 PM, VAN RAAIJ , MARK JOHAN < [log in to unmask]> wrote: > We once had a more-or-less MBP-sized fragment before cleavage, but this > turned to be a spontaneous mutation. This expression experiment had been > started from a glycerol stock with an unknown number of growth cycles prior > to expression. > Starting from a fresh transformation with the purified and sequenced > plasmid solved the problem. > Since then, I insist everybody does a fresh transformation before every > expression experiment and not generate extra growth/dilution cycles beyond > the normal transformation, growth on plate, overnight culture, dilution > into large-scale expression culture. > > > > Quoting "Bosch, Juergen": > > @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 >> >> >> >> >> >> > > > Mark J van Raaij > Laboratorio M-4 > Dpto de Estructura de Macromoléculas > Centro Nacional de Biotecnología - CSIC > c/Darwin 3, Campus Cantoblanco > 28049 Madrid > tel. 91 585 4616 > email: [log in to unmask] >