I am trying to measure differences in eNOS activity in skeletal muscle in sedentary and exercised mice, following the conversion of 3H-arg to 3H-cit, and assaying the samples with and with out L-NAME to get eNOS activity. However, I am having multiple problems, from signal no different from the blank (even with ~300 ug protein, fresh 1.25 mM L-NAME, fresh buffer components (including FAD, FMN, BH4, NADPH, CaM) and plenty of CaCl2), to no difference in activity with and without L-NAME, to huge variance between replicates (my pipetting is not that bad!!). I have 10 uM Arg total (1 uM 3H-Arg) in the assay, and allowing the samples to react for 30 min at 37C. I stop the reaction with NaAcetate pH 5.5 / 5 mM EDTA, and then pass the solution over dowex AG 50W x8 (Na form) to bind unreacted Arg in the solution. I am happy to share my protocol with anyone who can help me figure out what I am doing wrong!! Thanks for any and all help!
Hi Adele,
You may try to remove myoglobin from your skeletal muscle extracts, which tends to interfere with many enzymatic reactions. My 2 cents...
Gedas
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