Signaling Pathway Assays

pNPP Phosphatase Assay Kit (BA0167)

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SKU:
BA0167
Product Type:
Assay
Research Area:
Metabolism
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Description

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pNPP Phosphatase Assay Kit (BA0167) (BA0167)

The pNPP Phosphatase Assay Kit (SKU: BA0167) provides a colorimetric method for determining phosphatase activity at 405 nm. Para-nitrophenyl phosphate (pNPP) is a chromogenic substrate for most phosphatases, including alkaline phosphatases, acid phosphatases, protein tyrosine phosphatases and serine/threonine phosphatases, yielding para-nitrophenol, which becomes an intense yellow soluble product under alkaline conditions. This homogeneous mix-and-measure assay involves simply adding a single reagent to the phosphatase and measuring product formation on an absorbance reader. The assay can be performed in cuvettes, tubes or multi-well plates at either room temperature or 37 C and completed within 30 minutes. It offers high sensitivity, with a detection limit generally of 3 ng phosphatase or below, and is well suited to high-throughput inhibitor screening.

Product Name:pNPP Phosphatase Assay Kit (BA0167)
SKU:BA0167
Detection Method:Colorimetric
Detection Range:Detection limit generally 3 ng phosphatase or below
Sample Type:Purified phosphatase enzyme preparations
Species Reactivity:All
Assay Time:10 - 30 minutes
Kit Size:500 Assays
Equipment Required:Microplate reader
Storage:Store the Reagent at -20 C and all other components at 4 C.
Shelf Life:12 months after receipt
Shipping:Room Temperature

A colorimetric assay for the determination of phosphatase activity at 405 nm using the chromogenic substrate pNPP. Phosphatases release para-nitrophenol, which forms an intense yellow product under alkaline conditions. The homogeneous mix-and-measure format requires no wash or transfer steps and is compatible with high-throughput liquid-handling instruments.

  • High sensitivity and a wide linear range, with a detection limit generally of 3 ng phosphatase or below
  • Homogeneous, simple procedure with no wash or transfer steps
  • Completed within 30 minutes
  • Robust and amenable to high-throughput screening
  • Enzyme activity assay and quality control for phosphatase production
  • Characterisation of the kinetics of phosphatase reactions
  • High-throughput screening for phosphatase inhibitors

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1Equilibrate all reagents to room temperature for 30 min. Prepare enough pNPP Substrate by mixing 0.5 uL Reagent and 50 uL Assay Buffer per assay.
2Serially dilute the enzyme in a suitable enzyme buffer and transfer 50 uL of each dilution into wells of a clear flat-bottom 96-well plate, including a blank containing 50 uL enzyme buffer only.
3Initiate the reaction by adding 50 uL pNPP Substrate to each well.
4Incubate for 10-30 min at room temperature (or 37 C).
5Stop the reaction by adding 50 uL Stop Solution and mix by tapping the plate or shaking briefly.
6Read the absorbance at 405 nm. A 384-well format is also supported using 25 uL of each reagent.

Average the triplicate readings and subtract the blank. Calculate enzyme activity from the Beer-Lambert law as Enzyme activity (umoles/min/ug) = (V x OD405) / (epsilon x incubation time x enzyme), where epsilon is 1.78 x 10^4 M^-1 cm^-1 for para-nitrophenol, V is the final assay volume (150 uL for 96-well, 75 uL for 384-well) and OD405 is divided by the light-path length. The turnover number equals enzyme activity multiplied by the enzyme molecular weight.

ComponentQuantityStorage
Reagent280 uL-20 C
Assay Buffer25 mL4 C
Stop Solution25 mL4 C
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