I'm now setting the protocol for measuring in vitro cytochrome P450 activity
using protocol from Thomas Van Leeuwen.Article Comparative acaricide susceptibility and detoxifying enzyme ...
I've used whole body protein by simply grinding the whole organism of honey bee,
and used boiled protein for the control but it hadn't worked -
there were no difference between control and experimental group.
1) Should I have to extract the microsome and use it to measure the CYP450 activity?
2) Can I get any useful advice on measuring cytochrome P450?
I think you would be more likely to get useful results by working with microsomes rather than crude bee extract because the specific activity will be much higher and there will be less interference from other material in the extract.
There are many enzymes in crude extracts that could interfere with assays.
Microsomes, or post mitochondrial fraction may give you better material for assays.
Please see this link for a useful advise on measurement of P450. It is quite detailed. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843963/
Then, bubble some CO into it to see if it’s inhibitable &/OR try some classic P450 inhibitors in the subcellular fractions.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes