35S labeling E coli

[Michael Benedik]

In article <pablo-061094162048 at 128.42.239.32>, pablo at bioc.rice.edu (Paul Campbell) writes:
>I need to 35S label (met and cys) my favorite protein that I have in a
>pET21 vector (IPTG-inducible), but I'm not sure how to do it. So, I've got
>two questions:
>	 1.  Should I add the label before or after I induce expression of my
>favorite
>      protein with IPTG?
>	 2.  Do I have to use a synthetic medium without met and cys, or can I use
>      a rich medium like LB, 2xYT or TB?
>
>Any and all help would be appreciated.
>Thanks a million.
>
>Paul Campbell (a.ka. pablo at bioc.rice.edu)
>Dept. Biochemistry
>Rice University


Add label after induction. The pool of S-35 is depeleted pretty quickly
so you only get effective labelling for a short while.

You can label in LB, but not with as high efficiency as minimal media
lacking met and cys. We have done it pretty often for quick screening and
it does work.


**********************************************************************
Michael Benedik						benedik at uh.edu
Department of Biochemical Sciences
University of Houston