pNPP Phosphatase Assay Kit

Catalog No. ABIN1000337

Product Details

Target: pNPP Phosphatase

Application: Activity Assay (AcA)

Characteristics: High sensitivity and wide linear range. The detection limit is generally 3 ng 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 high- throughput liquid handling instruments.

Application Details

Application Notes: Enzyme Activity Assay and Quality Control for phosphatase production. Characterization of Kinetics of phosphatase reaction. Drug Discovery: high-throughput screen for phosphatase inhibitors.

Comment:

(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 100 mM sodium acetate (pH 5.5), 10 mM MgCl2 as Enzyme Buffer. For an alkaline phosphatase, we recommend using our DALP-250 Assay Kit.

Protocol: 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 0.5 µL Reagent and 50 µL Assay Buffer.
2. Serially dilute enzyme in a proper Enzyme Buffer. Prepare enough solution for triplicate assays. Transfer 50 µL of each enzyme dilution to wells of a clear, flat-bottom 96-well plate. In addition, prepare a blank control that contains 50 µL Enzyme Buffer only. Initiate the reaction by adding 50 µL pNPP Substrate to each well.
3. Incubate for 10-30 minutes at room temperature.
4. Stop the reaction by adding 50 µL 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.

Restrictions: For Research Use only

Handling

Storage: 4 °C

References

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Olsen, Sarras, Intine: "Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus." in: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, Vol. 18, Issue 5, pp. 532-42, (2010) (PubMed).

Thanissery, McReynolds, Conner, Macklin, Curtis, Fasina: "Evaluation of the efficacy of yeast extract in reducing intestinal Clostridium perfringens levels in broiler chickens." in: Poultry science, Vol. 89, Issue 11, pp. 2380-8, (2010) (PubMed).

Lee, Jeong, Han, Lee, Phee, Hahn, Ronald: "The Xanthomonas oryzae pv. oryzae PhoPQ two-component system is required for AvrXA21 activity, hrpG expression, and virulence." in: Journal of bacteriology, Vol. 190, Issue 6, pp. 2183-97, (2008) (PubMed).

Nakano: "Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivity." in: The British journal of dermatology, Vol. 156, Issue 5, pp. 848-60, (2007) (PubMed).

Monick, Powers, Gross, Flaherty, Barrett, Hunninghake: "Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase 1." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 177, Issue 3, pp. 1636-45, (2006) (PubMed).

Sawaguchi, Ohkubo, Matsubara: "Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri." in: Zoological science, Vol. 23, Issue 11, pp. 1021-9, (2006) (PubMed).

Target Details

Target: pNPP Phosphatase

Background: Quantitative determination of phosphatase activity by colorimetric (405nm) method.

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. phosphatase p-Nitrophenyl phosphate p-nitrophenol + phosphate This homogeneous mix-and-measure assay involves simply adding a single reagent to the phosphatase and measuring the product formation on an absorbance reader. The assay can be conveniently performed in cuvettes, tubes or multi-well plates at either room temperature or 37° C.