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: [EMAIL PROTECTED]
*******************************************
----- Original Message -----
From: <[EMAIL PROTECTED]>
To: <[email protected]>
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
