35S labeling E coli

[Arthur Hunt]

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,
   A long time ago (15 yrs), I made some labeled E. coli glutamine binding
protein by adding 25 mCi of 35S-SO4 to a liter of minimal medium E.  This
worked great, as I recall (good yield, high specific activity, low cost).
If you can induce your protein in minimal media, I think this would give you
good results (I forget what the kinetic constants for met and cys transport
in E. coli are, but these numbers are important to keep in mind if you are
going to add met and cys instead of SO4).  Adding the label right before in
induction would probably be a good idea as well (I didn't have to worry about
this in my experiments).
Good luck.
 
Arthur Hunt
Dept. of Agronomy
University of Kentucky
Lexington, KY 40546-0091