p-Nitrophenyl Phosphate (pNPP, KR-pT-IRR), Phosphatase Assay Kit, BioAssay(TM)

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.\n\nKey Features:\nHigh 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.\nRobust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.\n\nApplications:\nEnzyme Activity Assay and Quality Control for phosphatase production.\nCharacterization of Kinetics of phosphatase reaction.\nDrug Discovery: high-throughput screen for phosphatase inhibitors.\n\nKit Contents:\nCatalog # Size (assays) Reagent Assay Buffer Stop Solution\nXXXX-XXX 500 280ul 25ml 25ml\nXXXX-XXX 1,000 550ul 50ml 50ml\nXXXX-XXX >10k customized customized customized\nStorage conditions. The kit is shipped at ambient temperature. Store Reagent at -20 degrees C and other components at 4 degrees C. Shelf life: 12 months after receipt.\nPrecautions: 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.\n\nProcedure Using 96-Well Plate\n1. 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.\n2. 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.\n3. Incubate for 10-30 minutes at room temperature.\n4. 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.\n5. Read the absorbance of each well at 405 nm.\n\nProcedure Using 384-Well Plate\nThe 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.\n\nGeneral Considerations:\n(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 degrees C. (2). Assays can be performed at room temperature or at 37 degrees 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(TM).\n\nData Analysis:\nCalculate 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

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SPECIFICATIONS

Catalog Number

N2600-37

Size

1Kit

References

1. Monick, M.M., et al., (2006). Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase. J. Immunol. 177: 1636