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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
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.