 |
 |
Rana, It is hard to answer you question without more details (MW and pI of your target protein). MBP binds very well to amylose resins and is usually quite easily bound to anion exchange resins. Did you just run a "standard" ion exchange protocol or try different pH regimes? However, you did mention you have used a detergent. Why do you do that? MBP binding to amylose resins can be markedly disturbed in the presence of several different detergents, which is a particularly bad thing for a membrane protein fusion. That is why all our MBP fusion constructs have an additional His-tag. If you really don't need the detergent, leave it out, then try the amylose resin again. One other point is how old is your amylose resin and, if you express in E. coli, do you regularly add a little glucose to the media? Amylose resin is degraded by E. coli amylases, which I believe is suppressed when glucose is in the medium. However, inferring from your email, I would suppose that you initially purified the MBP fusion using the amylose resin. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [log in to unmask] **************************************************************** On Mar 27, 2014, at 6:49 AM, rana ibd <[log in to unmask]> wrote: > Dear Mark > Thank you for yor reply, and yes I have tried adding it to the maltose resin after cleavage but the MBP runs through with my protein, I have also tried 1M NaCl but with no luck and I also apply detergent after cleavage to the dialysis buffer because I usually dialyze after cleavage , is there anything that could maybe precipitate the MBP > Best Regards > Rana > > > From: Mark J van Raaij <[log in to unmask]> > To: rana ibd <[log in to unmask]> > Sent: Thursday, March 27, 2014 11:35 AM > Subject: Re: [ccp4bb] maltose binding protein > > did you try the maltose-resin? > in principle it should bind MPB but not your protein. You can try to add salt or detergents to disturb interaction between MBP and your protein (also in gel filtration). > No guarantee of success, unfortunately not all protein behave "nicely". > > Mark J van Raaij > Lab 20B > 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 27 Mar 2014, at 11:26, rana ibd wrote: > > > Dear CCP4 > > Does anyone know how to remove the maltose binding protein after cleavage from the target protein; I have tried gel filtration and ion exchange but with no luck, my protein is interacting with the MBP even after complete cleavage. I would be grateful for any help or suggestions > > Best Regards > > Rana > >