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.

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.

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.

Characterization of Kinetics of phosphatase reaction.

Drug Discovery: high-throughput screen for phosphatase inhibitors.

Storage conditions. The kit is shipped at ambient temperature. Store Reagent at -20°C and other components at 4°C. Shelf life: 12 months.

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.

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.

The 384-well assay procedure is the same as for the 96-well plate protocol except that 25 μL is mixed with 25 μL pNPP Substrate. After the incubation, add 25 μL Stop Solution.

(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.

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., 150 μL for 96-well plate assay and 75 μL for 384-well plate assay.

Left: PTP1B is one member of the large protein tyrosine phosphatase family. The pNPP assay was performed in a 384-well plate. After the reaction was initiated by the addition of pNPP Substrate, the plate was read on every 0.5 minutes for 30 minutes.

Right: a plot of the initial rate (Vo) against enzyme concentration. The detection limit was 3 ng PTP1B. The enzyme activity measured from the linear range was 2.2 μmoles/min/μg. The turn-over number was 82,280 per minute.

1. Monick, M.M. et al (2006). Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase. J. Immunol. 177: 1636–1645.

2. Nakano, Y. (2007). Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivity. British J. Dermatology 156 (5): 848–860.

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.

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.

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.

Characterization of Kinetics of phosphatase reaction.

Drug Discovery: high-throughput screen for phosphatase inhibitors.

Storage conditions. The kit is shipped at ambient temperature. Store Reagent at -20°C and other components at 4°C. Shelf life: 12 months.

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.

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.

The 384-well assay procedure is the same as for the 96-well plate protocol except that 25 μL is mixed with 25 μL pNPP Substrate. After the incubation, add 25 μL Stop Solution.

(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.

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., 150 μL for 96-well plate assay and 75 μL for 384-well plate assay.

Left: PTP1B is one member of the large protein tyrosine phosphatase family. The pNPP assay was performed in a 384-well plate. After the reaction was initiated by the addition of pNPP Substrate, the plate was read on every 0.5 minutes for 30 minutes.

Right: a plot of the initial rate (Vo) against enzyme concentration. The detection limit was 3 ng PTP1B. The enzyme activity measured from the linear range was 2.2 μmoles/min/μg. The turn-over number was 82,280 per minute.

1. Monick, M.M. et al (2006). Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase. J. Immunol. 177: 1636–1645.

2. Nakano, Y. (2007). Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivity. British J. Dermatology 156 (5): 848–860.

Phosphate (Pi) is one of the most important ion species in nature. Phosphate is present in all biological systems. It is a major constituent in minerals and fertilizers, and is a component of industrial wastewater. Thus accurate determination of phosphate concentration finds numerous  applications in pharmacology, biomedical research, clinical chemistry, industrial process monitoring and environmental monitoring. Simple, direct and automation-ready procedures for measuring phosphate concentration in biological and environmental samples are becoming popular. BioAssay Systems' phosphate assay kit is designed to measure phosphate ion directly in samples without any pretreatment. The improved Malachite Green method utilizes the malachite green dye and molybdate, which forms a stable colored complex specifically with inorganic phosphate. The intensity of the color, measured at 620nm, is directly proportional to the phosphate concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples.

Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 30 min. Can be readily automated as a high-throughput assay for thousands of samples per day.

Improved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Assays can be executed in cuvet or 96-well plate.

Low interference in biological samples. No pretreatments are needed. Assays can be directly performed on raw biological samples i.e., in the presence of lipid, protein and minerals.

Drug Discovery/Pharmacology: effects of drugs on Pi metabolism.

Food and Beverages: Pi determination.

Environment: Pi determination in water, soil and fertilizer.

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.

Important: bring reagents to room temperature and shake before use.

1. Set up standards and samples. Transfer 50 μL distilled water ("Blank"), Standard and samples in duplicate wells of a clear bottom 96-well plate.

2. Add 100 μL Reagent and tap lightly to mix.

3. Incubate 30 min at room temperature and read optical density at 620nm (600-660nm).

1. Set up test tubes labeled Blank, Standard, Samples. Transfer 400 μL Water, Standard and samples to appropriately labeled tubes.

2. Add 800 μL Reagent and tap lightly to mix.

3. Incubate 30 min at room temperature, transfer to cuvet and read optical density at 620 nm (600-660nm).

Important: (1) if sample OD is higher than the OD for standard, dilute samples in distilled water and repeat the assay. (2) It is not necessary to prepare a calibration curve, because the concentration of the provided standard lies within the linear range. (3) Precipitation may occur at high concentrations of phosphate (>100 μM), or in the presence of high concentrations of e.g. proteins and metals. In this case, dilute samples in distilled water and repeat the assay.

The phosphate concentration of Sample is calculated as

ODBLANK, ODSTANDARD and ODSAMPLE are OD620nm values of Blank, Standard and Sample, respectively.

Conversions: 1 mg/dL Pi equals 105.3 μM, 0.001% or 10 ppm.

Pipeting devices and accessories.

Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.

Spectrophotometer and cuvets for measuring OD 620nm.

Biological Samples: 1. Commercial 2%  reduced fat milk (Kirkland). 2. Invitrogen fetal bovine serum. 3. Fresh human urine. Water samples: 4. Tap water (Hayward, CA). 5. Tap water (San Bruno, CA). Food and Beverages: 6. Crystal Geyser natural alpine spring water. 7. Coca-cola® classic coke. 8. Lipton Lemon iced tea. Environmental: 9. Soil extract. 5.6 g of soil (Hayward, CA) was extracted with 10 mL MilliQ water. The supernatant was centrifuged to remove any insoluble particles. Clear supernatant was assayed.

1. Abranches, J. (2008). CcpA regulates central metabolism and virulence gene expression in Streptococcus mutans. J Bacteriol. 190(7):2340-9.

2. Hildebrand, J. et al (2009) Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout. Comp Biochem Physiol C Toxicol Pharmacol. 149(1):65-72.

The PiBlueTM Phosphate Assay Kit is based on a proprietary formulation of the malachite green dye. The PiBlueTM reagent forms a blue colored complex with free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 - 660 nm) or on a plate reader. The non-radioactive colorimetric assay kits have been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be performed in tubes, cuvettes or multi-well plates. The assays can be conveniently executed in 96-well plates for highthroughput screening of enzyme inhibitors.

High sensitivity and wide detection range: detection of as little of 20 pmoles of phosphate and useful range between 0.4 μM and 50 μM phosphate.

Fast and convenient: single reagent “mix-and-measure” assay allows quantitation of free phosphate within 30 minutes.

Compatible with routine laboratory and HTS formats: assays can be performed in tubes, cuvettes or microplates, on spectrophotometers and plate readers.

Robust and amenable to HTS: Z’ factors of 0.7 to 0.9 are observed in 96- well plates. Can be readily automated on HTS liquid handling systems.

Lipase Assays: liberation of phosphate from phospholipids

Nucleoside Triphosphatase Assays: liberation of phosphate from nucleoside triphosphates (ATP, GTP, TTP, CTP etc).

Quantitation of Phosphate in phospholipids, proteins and DNAs, etc.

Drug Discovery: high-throughput screen for phosphatase inhibitors.

Storage conditions. The PiBlueTM Reagent and standard is stable for 12 months when stored at 4°C.

Precautions: reagents are for research use only. This reagent contains 0.44 M sulfuric acid. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

2. Transfer 50 μL test sample (e.g. enzyme reaction) in duplicate into wells of the microplate. In the case of enzyme reactions, the reaction may be terminated by either adding a specific inhibitor, or can be stopped directly by the addition of the PiBlueTM Reagent. Reaction buffer can be added as a blank control for the samples.

3. Add 100 μL of the PiBlueTM Reagent to each well. Mix by tapping the plate.

4. Incubate for 30 min at room temperature for color development.

5. Measure absorbance at 620 nm (600 nm - 660nm) on a plate reader.

For cuvette assays, add 800 μL Reagent to 400 μL sample and standards. Perform the assay as described for the microplate assay.

Precipitation may occur at high concentrations of phosphate (>100 μM), or in the presence of high concentrations of e.g. proteins and metals. If precipitation occurs, dilute samples in distilled water and repeat the assay.

Enzyme reaction buffer. Because any exogenous free phosphate would interfere with the assay, it is important to ensure that the protein preparation, the reaction buffer and lab wares employed in the assay should not contain free phosphate. This can be conveniently checked by adding the Reagent to the buffer and measuring the color formation.

Plot pmoles phosphate versus OD620nm for the standard curve. Use linear regression analysis to determine amount of free phosphate in the test samples.

1. Rumsfeld J, Ziegelbauer K, Spaltmann F (2000). High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example. Protein Expr Purif. 18(3):303-9.

2. Cogan EB, Birrell GB, Griffith OH (1999). A robotics-based automated assay for inorganic and organic phosphates. Anal Biochem. 271:29-35.

3. Ng DH, Harder KW, Clark-Lewis I, Jirik F, Johnson P (1995). Nonradioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells. J Immunol Methods. 179(2):177-85.

4. Fisher DK, Higgins TJ (1994). A sensitive, high-volume, colorimetric assay for protein phosphatases. Pharm Res. 11(5):759-63.

The Malachite Green Phosphate Assay Kit is based on quantification of the green complex formed between Malachite Green, molybdate and free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 - 660 nm) or on a plate reader. The non-radioactive colorimetric assay kits have been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be executed in tubes, cuvettes or multi-well plates. The assays can be conveniently performed in 96- and 384-well plates for high-throughput screening of enzyme inhibitors.

High sensitivity and wide detection range: detection of as little of 1.6 pmoles of phosphate and useful range between 0.02 μM and 40 μM phosphate.

Fast and convenient: homogeneous “mix-and-measure” assay allows quantitation of free phosphate within 20 minutes.

Compatible with routine laboratory and HTS formats: assays can be performed in tubes, cuvettes or microplates, on spectrophotometers and plate readers.

Robust and amenable to HTS: Z’ factors of 0.7 to 0.9 are observed in 96- well and 384-well plates. Can be readily automated on HTS liquid handling systems.

Lipase Assays: liberation of phosphate from phospholipids

Nucleoside Triphosphatase Assays: liberation of phosphate from nucleoside triphosphates (ATP, GTP, TTP, CTP etc).

Quantitation of Phosphate in phospholipids, proteins and DNAs, etc.

Drug Discovery: high-throughput screen for phosphatase inhibitors.

Storage conditions. The reagents and standard are stable for one year when stored at 4°C.

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.

2. Transfer 80 μL test samples into separate wells of the plate.

Note: in the case of enzyme reactions, the reaction may be terminated by adding a specific inhibitor, or can be stopped directly by the addition of the Working Reagent. Dilution of reaction mixture may be necessary prior to the assay (see General Considerations). For ATPase or GTPase assays, the ATP or GTP concentration should be lower than 0.25 mM. If the reaction mixture contains > 0.25mM ATP or GTP, dilute samples in distilled water. For example, if the ATPase reaction contained 1 mM ATP, at the end of reaction dilute reaction mixture 4-fold in water prior to the assay.

3. Add 20 μL of Working Reagent to each well. Mix gently by tapping the plate.

4. Incubate for 30 min at room temperature for color development.

5. Measure absorbance at 600 nm - 660nm (620 nm) on a plate reader. For assays in 384-well plates, the procedures are the same, except that the volume of the standard and sample solution should be 40 μL and that of the Working Reagent should be 10 μL.