I agree with Mark, except that I wouldn't even try sonication, Triton or freeze/thaw cycles in that case.
I'd look for emulsification (with a Homogenizer) in a cold room, but if you go quickly and gently with the French Press (either in a cold room or by using a cold piston) it might help. Don't use too much pressure, it heats up the sample. I also agree that if it migrates as a single peak in gel filtration and in heparin sepharose, there is no reason for not setting some drops with it. And if you decide to do it, then simplify the purification and avoid submitting the protein to treatments that are not helping to get it purer. (I just found it weird that your "fraction 6" has a huge load of protein , I guess those are actually the beads from the purification or something like that? In any case it seems to me that the fraction volume could be increased) Carlos Em 15/02/2012, às 16:39, Mark J van Raaij escreveu: > try experimenting with different, especially protease-deficient, E coli > strains to express the protein and try different methods to lyse the bacteria > (sonication, french-press, emulsification, bead-beater, mortar & pestle under > liquid nitrogen). > > on the other hand, if you are lucky, you are just proteolysing some surface > loops and can still purify and crystallise the protein. This was done on > purpose for the cap-binding complex, see: > Crystal structure of the human nuclear cap binding complex. > Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S. > Mol Cell. 2001 Aug;8(2):383-96. > > Mark J van Raaij > Laboratorio M-4 > Dpto de Estructura de Macromoleculas > Centro Nacional de Biotecnologia - CSIC > c/Darwin 3 > E-28049 Madrid, Spain > tel. (+34) 91 585 4616 > http://www.cnb.csic.es/~mjvanraaij > > > > On 15 Feb 2012, at 14:09, Sivasankar Putta wrote: > >> Dear All, >> >> Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi >> domain) DNA binding protein, that we are expressing at 18 degree Centigrade >> in E. Coli. The protein appears to degrade during purification; we have >> protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM >> EDTA and 1mM DTT throughout during purification ( right from lysis stage). >> We handle the protein at 4 degree Centigrade. >> >> Can you please suggest what precautions we can try to avoid such degradation >> ? >> >> Please find the attached gel picture regarding protein >> >> Sivasankar Putta >> >> >> >> <proteingel.pdf>
