> ...Products like PEP, Pyruvate, Fructose-1,6-bisphosphate, ATP, NAD, NADP etc.
> I know how I can detect the isolated intermediate products, but I'm searching
> for a method to break the E.coli cells very fast...
We've been trying three methods, one of which involves rapid chilling with
ice and centrifugation, which probably won't work with your Gram negative
organism (intracellular metabolites will leak out). We also have tried
spinning culture samples through a layer of silicone into a small volume of
perchloric acid. You may also be able to layer culture onto a small amount
of chlorloform to get similar results, although we haven't tried this on a
large scale. Others have sucked culture through a filter (0.6-0.8 um
doesn't clog as much) then digested the cells off the filter with cold TCA
or perchloric.
I'd be very interested to hear how you are measuring F16BP and PEP. (We are
trying it with a fluorimeter). Could you email some details?
Daniel R. Bond
drb13 at cornell.edu
