Cytochrome P450 1A2 Fluorometric Activity Assay Kit

• Cat.No.: Kit-2126
• Product Overview: CYP1A2 Activity Assay Kit enables rapid measurement of native or recombinant CYP1A2 activity in biological samples such as liver microsomes. The assay utilizes a non-fluorescent CYP1A2 substrate that is converted into a highly fluorescent metabolite detected in the visible range (Ex/Em = 406/468 nm), ensuring a high signal-to-background ratio with little interference by autofluorescence. A selective CYP1A2 inhibitor is provided for determination of CYP1A2 activity in heterogeneous biological samples, where other CYP isozymes may contribute to substrate metabolism. The inhibitor displays greater than 20-fold selectivity for CYP1A2 over other CYPs, ensuring targeted inhibition. CYP1A2 specific activity is calculated by running parallel reactions in the presence and absence of the selective inhibitor and subtracting any residual activity detected with the inhibitor present. The kit contains a complete set of reagents sufficient for performing 50 sets of paired reactions (in the presence and absence of inhibitor).

Specification

• Size: 100 assays
• Applications: Rapid assessment of native/recombinant CYP1A2 activity in lysates or microsomal fractions prepared from tissues, cells.Screening of drugs and novel ligands for interaction with native/recombinant CYP1A2.
• Storage: Store kit at -20°C and protect from light. Briefly centrifuge all small vials prior to opening. Allow the CYP1A2 Assay Buffer to warm to room temperature prior to use. Read entire protocol before performing the assay procedure.3-CHC Standard: Reconstitute in 110 µl of DMSO and vortex until fully dissolved to yield a 5 mM stock solution. The 3-CHC stock solution should be stored at -20°C and is stable for at least 3 freeze/thaw cycles.CYP1A2 Inhibitor (α-naphthoflavone): Reconstitute in 110 µl of acetonitrile and vortex until fully dissolved to yield a 1 mM stock solution. The stock solution is stable for 2 months at -20°C. To obtain a 2.5 µM working solution of α-naphthoflavone (5X final concentration), add 5 µl of the 1 mM stock solution to 1995 µl of CYP1A2 Assay Buffer. The 2.5 µM working solution should be stored at -20°C and used within one week.NADPH Generating System (100X): Reconstitute with 220 µl CYP1A2 Assay Buffer, aliquot and store at -20°C. Avoid repeated freeze/thaw cycles and keep on ice while in use.β-NADP+ Stock (100X): Dissolve in 110 µl CYP1A2 Assay Buffer and vortex thoroughly to yield a 10 mM solution of NADP+ (100X stock). Store at -20°C, stable for at least 3 freeze/thaw cycles.CYP1A2 Substrate: Reconstitute with 110 µl anhydrous reagent-grade acetonitrile and vortex until fully dissolved to obtain a 5 mM stock solution. Store at -20°C. Allow the vial to warm to room temperature before opening and promptly retighten cap after use to avoid absorption of airborne moisture.Recombinant Human CYP1A2: Do not reconstitute until ready to use. Reconstitute with 230 µl CYP1A2 Assay Buffer and add 20 µl of NADPH Generating System (100X). Mix thoroughly to ensure a homogenous solution (the solution will have a slightly opaque, milky appearance), aliquot and store at -80°C. Avoid repeated freeze/thaw cycles and use aliquots within one month (the Recombinant Human CYP1A2 will lose approximately 10% activity per week when stored at -80°C). Thaw aliquots rapidly at 37°C and place on ice until use (thawed aliquots should be used within 4 hours).