P450 Demethylation Fluorescent Activity Kit

Documentation

Specifications

• Assay Type Activity Assay
• Sample Types Any P450 system or biological system producing demethylation product, Formaldehyde
• Assay Duration 45 Minutes
• Samples/Plate 41 in Duplicate
• Readout Fluorescent, 510 nm Emission / 450 nm Excitation
• Standard Curve
• Description

  Assay Principle: 

  The P450 Demethylation Fluorescent Activity Kit quantitatively measures demethylating P450 system activity in any P450 system or biological system producing demethylation product, Formaldehyde. The P450 Demethylation Fluorescent Activity Kit is an Activity Assay with a run time of 45 minutes. Please read the complete kit insert for more information before performing this assay.

  Use our provided Formaldehyde Standard to generate a standard curve for the assay. Pipette the standards or diluted samples into a black microtiter plate. Add NADPH and allow the P450 demethylase reaction to occur, incubating at 37°C for 15-60 minutes. Add DetectX® Formaldehyde Detection Reagent to each well tapping the plate to ensure sufficient mixing of reagents. Then incubate the mixture covered at 37°C for 30 minutes. The fluorescent generating reaction occurs between the Formaldehyde Detection Reagent and the Formaldehyde generated from the P450 demethylase reaction within the samples. 

  After the 30-minute incubation, use a plate reader to detect and record the generated fluorescent signal at 510nm. Use the intensity and the standard curve to calculate the P450 demethylase activity in the samples.

  Background:

  Cytochrome P450s are present in most tissues and have critical biological functions such as hormone synthesis and breakdown, cholesterol synthesis, and vitamin D metabolism. Cytochrome P450 enzymes also metabolize drugs and play a crucial role in developing new drug entities, as drug interactions commonly inhibit enzyme activities. The enzymes are a superfamily of heme-containing enzymes. They display tremendous diversity regarding substrate specificity and catalytic activity. They use exogenous and endogenous compounds as substrates in their reactions and form part of multicomponent electron transfer reactions. These enzymes are also highly investigated in the toxicology field as many CYP P450 enzymes break down toxins to prevent damaging build-up within the body.