P450 enzyme systems diluted in the supplied Assay Buffer provided or a typical 0.1M phosphate buffer at pH 7.4 are compatible with this assay.
P450 demethylating reaction conditions We have ensured the DetectX® P450 Activity Assay detects the activity of the 2B4, 2D6 and 3A4 P450 systems.
Below we have listed the conditions we used in validating this fluorescent detec- tion system and the ability to quantitate the formaldehyde produced by the Cyp 2B4 P450 enzy- matic reaction.
Typical Cyp 2B4 Enzyme Reaction To duplicate wells add 15 μL of P450 enzyme system (equivalent molar ratios of 2B4 P450, Cy- tochrome P450 Oxidoreductase, and Cytochrome b5 in a pre-sonicated 0.66 mg/mL DLPC solu- tion), followed by 75 μL of the supplied Assay Buffer and 5 μL of P450 substrate.
Seal the plate and incubate for 5 minutes at 37 °C prior to addition of 5 μL of the reconstituted supplied NADPH activator.
Seal the plate again and incubate for 15 minutes at 37 °C.
Add 5 μL of the supplied Stop Solution followed by the addition of 25μL of the FDR to each well.
Reseal the plate and incubate at 37 °C for 30 minutes.
For calibration purposes to formaldehyde, the 15 μL of P450 enzyme solu- tion is replaced with standards made from the supplied Formaldehyde stock.
For calibration purposes to formaldehyde, the P450 enzyme solution is replaced with standards made from the supplied Formaldehyde stock. reaction overview P450 Reaction 1.
Carry out demethylating enzyme reaction. 2.
Stop the reaction (optimal), add FDR.
Formaldehyde Detection 3.
Incubate at 37 °C for 30 minutes, read signal. 4.
Calibrate to Formaldehyde generated.
The kit is unique in that the fluo- rescent substrate is not involved in the multicomponent P450 reaction, but measures the product of the demethylation, formaldehyde.
No separation or washing is required.
The kit has been validated for several P450 systems and should work with any biological system that is producing formaldehyde as a product of demethylation.
The kit provides an optimized buffer for P450, lyophilized vials of the cofactor, NADPH, for the reaction, a stable formaldehyde standard, the Formaldehyde Detection Reagent (FDR) and two 96 well plates for detecting the generated fluorescent signal.
The end user will have to provide the microsomal, baculosome system or the recombinant P450, reductase and cytochrome b5 system and any cofactors, etc. necessary for activity, along with any candidate drugs, inhibitors or activators being tested.
The reaction should be carried out in our supplied buffer or a similar PBS based buffer system.
Following the P450 NADPH-induced reaction, the generation of formaldehyde can be stopped by addition of a suitable inhibitor, or the supplied stop solution of acetic acid.
The FDR is then added to all the wells.
If calibration to formaldehyde is needed (for cross lab comparisons) then a formaldehyde standard curve generated from the supplied standard should be run.
After a short incubation at 37 °C for 30 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 450 nm.
The P450 activity is determined based upon formaldehyde production.
We have provided two 96 well plates for measurement but this assay is adaptable for higher density plate formats.
If substituting their own plates, the end user should ensure that their black HTS plate is suitable for use with these reagents prior to running samples.
Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all stan- dards and samples be run in duplicate to allow the end user to accurately determine P450 activity.
Ensure that all samples have reached optimal temperature for the P450 reaction and have been diluted as appropriate prior to running them in the kit.
NADPH Preparation Remove a vial of NADPH from the desiccator and add 600 μL of the Assay Buffer to the vial and vortex thoroughly.
Store any unused reconstituted NADPH at ≤ -20 °C for no more than 2 weeks.
Formaldehyde Standard Preparation Label six glass test tubes as #1 through #6.
Pipet 400 μL of Assay Buffer into tube #1 and 250 μL into tubes #2-#6.
Add 100 μL of the Formaldehyde stock solution to tube #1 and vortex com- pletely.
Add 250 μL of tube #1 to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #6.
The concentration of formaldehyde in tubes 1 through 6 will be 400, 200, 100, 50, 25, and 12.5 μM.
Use all Standards within 2 hour of preparation.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Buffer Volume (μL) 400 250 250 250 250 250 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μL) 100 250 250 250 250 250 Final Conc (μM) 400 200 100 50 25 12.5
- P450 reaction volume should be no more than 100 μL in each well including all cofactors, inhibitors and activators so that 25 μL of FDR can be added to each well for detection.
2. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. P450 Reaction
3. Pipet 95 μL of Assay Buffer as a Zero standard, standards or samples including all cofactors, substrates and/or inhibitors into the duplicate wells in the black plate. Seal with the plate sealer and incubate for 15 minutes at 37 °C.
4. Add 5 μL of the reconstituted NADPH to each well, seal the plate and incubate at 37 °C for 15-60 minutes (incubation time varies and is based upon the system and microsomes used - see pages 7 and 13).
5. Add 5μL of Stop Solution to each well. Formaldehyde Detection
6. Add 25 μL of the DetectX® Formaldehyde Detection Reagent to each well using a repeater pipet.
7. Gently tap the sides of the plate to ensure adequate mixing of the reagents.
8. Incubate at 37 °C for 30 minutes. Room temperature incubation will yield approximately 75 % of the fluorescent signal generated with 37 °C incubation.
9. Set plate parameters for a 96-well Corning Costar 3694 plate. See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data. Read the fluorescent signal from each well in a plate reader capable of reading the fluorescent signal at 510 nm with excitation at 450 nm. Please contact your plate reader manufacturer for suitable filter sets. This assay requires a plate reader with efficient fluorescence optics. Please refer to page 6 for details on increasing sensitivity.
Calculation of Results
Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard.
The sample activity obtained should be multiplied by the dilution fac- tor to obtain neat sample values.
For Research Use only