Microsomal incubationThe microsomal incubations that i did seem not to have worked. The final volume of the incubation mixture is 500 microlitre and consist of 25 microM of the test compound, 5mM of magnesium chloride, 1mM of NADPH, 10mM of sodium pyrophosphate, 50mM dipotassium orthophosphate buffer(pH=7.4) and 0.5mg/ml of human liver microsomes. Reaction was initiated by adding 50 microlitre of NADPH after pre-incubation for 5 minutes at 37 degrees celcius in a shaking water bath (200 ). The mixture was incubated for 60 minutes. does anyone know what might be wrong with this protocol?
Hi Chukwunonso,
Zillion reasons might be mentioned. Your "test compound" was not a P450 substrate. If it was, a compound concentration was too low to saturate P450 active site. NADHP sample was outdated (check the date of expiration). Human liver microsomes were not kept properly, so P450 converted into inactive P420 (check the CO-dithionite differential spectra using extinction coefficient, 91 mM−1 · cm−1). Your incubation time looks extremely long, as most monooxygenase reactions obey Michalis-Menten kinetics not longer than for 15-20 min of incubation time. If longer, the product formed might be degraded or involved in the second phase metabolism.
Best wishes,
Ilya
Thank you for your kind response ilya.
The test compound is largely a P450 substrate and the NADPH is not expired. The Microsomes are not degraded yet as we stored them at exactly -80. I certainly agree that the incubation time might be too long as the test compound mostly induces the activity of the CYP isoform involved in its metabolism so products might be formed and be degraded rather fast. I will adjust my incubation time and see what happens.
Thank you.
Chukwunonso
I have reduced the incubation time to 30 mins yet it seems not to have worked.
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