P450 Demethylation Activity Kit

P450 Demethylation

Application Kit

AA

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.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Demethylating P450 systems: liver microsomes or cerosomes such as Cyp P450 3A4, 2B4 and 2D6.

2°C to 8°C