P450 Demethylation Activity Kit
Cat# OKAU00010
Size : 2plate
Brand : Aviva Systems Biology
P450 Demethylation Activity Kit (OKAU00010)
| Datasheets/Manuals | Printable datasheet for P450 Demethylation Activity Kit (OKAU00010) |
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| Application | AA | ||||||||||||||||
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| ELISA Kit Principle | The DetectX® P450 Activity kit is designed to quantitatively measure the enzymatic activity of formaldehydeproducing enzymes such as Cytochrome P450s. The kit is unique in that the fluorescent 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 37C 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. | ||||||||||||||||
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| Additional Information | Background: The cytochromes P450 (P450s) are a superfamily of heme containing enzymes that display tremendous diversity with regard to substrate specificity and catalytic activity. P450s use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multicomponent electron transfer reactions (see figure). Catalysis by the eukaryotic P450 enzymes involves a multistep reaction cycle that includes two steps in which electron transfer is accomplished from a redox partner. The diflavin protein, NADPH cytochrome P450 reductase (reductase) contains both FAD and FMN and can transfer both electrons needed for the catalytic cycle. In some P450 reactions, the second electron of the reaction cycle also can be delivered by cytochrome b5. The P450 enzymes and cofactors of the mammalian drug-metabolizing system are embedded in the membrane of the endoplasmic reticulum. The P450s play a crucial role in the development of new drug entities as drug-drug interactions commonly arise from the inhibition of cytochrome P450 activities. Lipid plays an important role in the reconstitution of P450-dependent activities after protein purification. Most in vitro studies for the reconstitution of P450 activities use dilaurylphosphatidylcholine (DLPC) as the lipid component. The reconstitution of enzymatic activity involves a concentrated incubation of P450, its redox partners (NADPH and reductase), and lipid followed by dilution into the final assay components. The reported preincubation conditions vary significantly. | ||||||||||||||||
| Reconstitution and Storage | 2°C to 8°C | ||||||||||||||||
| Sample Type | Demethylating P450 systems: liver microsomes or cerosomes such as Cyp P450 3A4, 2B4 and 2D6. |
| Gene Full Name | P450 Demethylation |
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