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S35 labeling of E.coli prot: protocols?

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Fri Dec 1 15:45:26 EST 1995

In article <49nk9s$8dc at pulp.ucs.ualberta.ca>, cfisher at gpu5.srv.ualberta.ca
(Carl Fisher) wrote:

Hi Carl,

When we are metabolically labelling E.coli BL21(DE3)/T7 plasmid, we grow
in M9 minimal medium + 0.2% casamino acids + 0.6% glycerol + antibiotics
until the OD600 is 0.4-0.6, add 35S-met to 5u Ci/ml, add IPTG to 0.4-1mM,
incubate for 30 minutes, add rifampicin to 400 ug/ml and continue
incubation. Vector-only control is run in parallel. Take 100 ul aliquots
at time-points, harvest cells and boil in 50 ul 2X SDS-PAGE buffer for 5
minutes. Use 5-10 ul lysate per lane for PAGE screening.


Karl "different spelling" Fischer

Karl Fischer
tyr-2 at bones.biochem.ualberta.ca

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