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Re: [ccp4bb] protein degradation? |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: protein degradation? From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU Date: 2007-11-04 One piece of advice for speeding up the purification process is to use a great trick I heard about from Michael Rout, who uses this to trap protein-protein complexes from yeast: Pellet your cells as firmly as possible. Go into a cold room, scoop the pellet into the back of a big syringe (10, 20mL, etc.), replace the plunger, and extrude the pellet material through a thin needle into liquid nitrogen. Rout said it comes out like spaghetti, but in my hands, it became little droplets. Anyway, now you have small, frozen "noodles" or "pills," which are not degrading at all, because they are frozen at -80degC. For the next step, Dr. Rout uses an industrial ball mill, but I have used a ordinary ceramic mortar and pestle partially immersed in a close-fitting styrofoam box of liquid N2 (this need not be in a cold room). Anyway, the pellets are put into the mortar and pestle and laboriously crushed until a fine powder, with a bit of liqN2 to assure the temp is really low. This grinding pretty much destroys all of the cells, and while I am forced to use this technique as I am working with yeast right now (for which other lyses do not work), I would use it for bacteria as well. The main benefit is that all the sonication or french pressing is a major opportunity for the proteases to go to work (in spite of inhibitors, in my experience), whereas using this method allows all of the lysis to be carried out without any proteolysis whatsoever. Furthermore, the resultant fine powder can be weighed out in whatever way is most convenient for test purifications, etc., and if sprinkled on the surface of the resuspension buffer, is instantly resuspended. The degree of lysis is, in my hands, excellent. I suppose the negative side of this is the grinding in the mortar, but for me, it is still better than shivering in the cold room by the sonicator. Regards, Jacob Keller ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: j-keller2@northwestern.edu ******************************************* ----- Original Message ----- From: To: Sent: Sunday, November 04, 2007 4:18 PM Subject: Re: [ccp4bb] protein degradation? > Some proteases are metal-dependent, and inhibitors for those aren't > Ni-column-compatible. "We" (meaning my students) found that it helps to > (1) work very quickly and (2) put EDTA into the fraction collector tubes > before eluting from the Ni column. > > > At 06:22 AM 11/4/2007, Vijay Kumar wrote: >>Hi, >> >>I have been trying purify a N-ter his-tagged protein over-expressed in >>E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS >>PAGE which are very close each other (top band in the right MW and more >>intense than the lower band). Western blot (for his-tag) of the gel gave >>signal for both the bands. Mass spec results confirmed both protein bands >>are the same. So I think it could be C-ter degradation of my protein. Also >>the 2 bands exist after ion-exchange and sizing column. >> >>I use commercially available complete protease inhibitor tablets >>(increasing concentration has no effect) and sonication for lysis. I am >>wondering if people have encountered the same problem and got any >>suggestions? >> >> >>Thanks in advance. >> >>Regards, >> >>Vijay > > --------------------------------------------------------------------------------------------------------------------------- > Phoebe A. Rice > Assoc. Prof., Dept. of Biochemistry & Molecular Biology > The University of Chicago > phone 773 834 1723 > fax 773 702 0439 > http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 > http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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