pgex expression

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Fri Mar 27 14:51:47 EST 1998


Mr. J Hinshelwood <jhinshel at hgmp.mrc.ac.uk> wrote:
> When I cleave my protein from the gst (using thrombin) on the GST-agarose
> I get a product that runs as a single band by SDS page plus a few minor
> contaminants. If I run this product on an IEF gel I see multiple bands and
> likewise if I try to purify this by ion exchange I get multiple peaks.
> Each of these peaks from the ion exchange run at the same hight on SDS
> PAGE and all are detected by my antibody in western blot.

Thrombin is not as specific as Pharmacia wants you to believe. If you
can, try to do mass spectroscopy with your protein. We had at least one
case where the thrombin chewed off a few amino acids in addition to
cleaving behind the GST. This can result in a change of the isoelectric
point which is detectable by ion exchange chromatography but not by
SDS PAGE.

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */



More information about the Proteins mailing list