Para-nitrophenyl phosphate (pNPP) is a chromogenic substrate for most phosphatases such as alkaline phosphatases, acid phosphatases, protein tyrosine phosphatases and serine/threonine phosphatases. The reaction yields para-nitrophenol, which becomes an intense yellow soluble product under alkaline conditions and can be conveniently measured at 405 nm on a spectrophotometer.||Key Features:|High sensitivity and wide linear range. The detection limit is generally 3ng phosphatase or below. Homogeneous and simple procedure. No wash or reagent transfer steps are involved. The assay can be completed within 30 minutes.|Robust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.||Applications:|Enzyme Activity Assay and Quality Control for phosphatase production.|Characterization of Kinetics of phosphatase reaction.|Drug Discovery: high-throughput screen for phosphatase inhibitors.||Kit Contents:|Catalog # Size (assays) Reagent Assay Buffer Stop Solution|XXXX-XXX 500 280ul 25ml 25ml|XXXX-XXX 1,000 550ul 50ml 50ml|XXXX-XXX >10k customized customized customized|Storage conditions. The kit is shipped at ambient temperature. Store Reagent at -20°C and other components at 4°C. Shelf life: 12 months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedure Using 96-Well Plate|1. Equilibrate all reagents to room temperature by allowing them to stand for 30 minutes at room temperature. Prepare enough pNPP Substrate by mixing per assay 0.5ul Reagent and 50ul Assay Buffer.|2. Serially dilute enzyme in a proper Enzyme Buffer. Prepare enough solution for triplicate assays. Transfer 50ul of each enzyme dilution to wells of a clear, flat-bottom 96-well plate. In addition, prepare a blank control that contains 50ul Enzyme Buffer only. Initiate the reaction by adding 50ul pNPP Substrate to each well.|3. Incubate for 10-30 minutes at room temperature.|4. Stop the reaction by adding 50ul Stop Solution. Mix by quickly tapping the plate. Alternatively, plates can be shaken for 10 seconds on an orbital plate shaker.|5. Read the absorbance of each well at 405 nm.||Procedure Using 384-Well Plate|The 384-well Assay Procedure: is the same as for the 96-well plate protocol except that 25ul is mixed with 25ul pNPP Substrate. After the incubation, add 25ul Stop Solution.||General Considerations:|(1). Fresh reconstitution of the Reagent is recommended although the reconstituted pNPP Substrate may be stable for up to 4 weeks when stored at -20°C. (2). Assays can be performed at room temperature or at 37°C. (3). The pH of the Assay Buffer is 7.2 and is compatible with the majority of neutral phosphatases such as protein phosphatases. For an acid phosphatase, we recommend using 100mM sodium acetate (pH 5.5), 10mM MgCl2 as Enzyme Buffer. For an alkaline phosphatase, we recommend using Alkaline Phosphatase Assay Kit, BioAssay™.||Data Analysis:|Calculate the average and standard derivations of the triplicate assays and subtract the blank values. Enzyme activity is calculated from Beer-Lambert law as follows, where e is the molar extinction coefficient (M-1 × cm-1). For p-nitrophenol, e=1.78 x 104 M-1 × cm-1. OD405nm (cm-1) is the absorbance at 405 nm divided by the light-path length (cm). V is the final assay volume, i.e., 150ul for 96-well plate assay and 75ul for 384-well plate assay.