Description
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.
| Step | Procedure |
| 1 | Equilibrate 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. |
| 2 | Serially 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. |
| 3 | Initiate the reaction by adding 50 uL pNPP Substrate to each well. |
| 4 | Incubate for 10-30 min at room temperature (or 37 C). |
| 5 | Stop the reaction by adding 50 uL Stop Solution and mix by tapping the plate or shaking briefly. |
| 6 | Read 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.
| Component | Quantity | Storage |
| Reagent | 280 uL | -20 C |
| Assay Buffer | 25 mL | 4 C |
| Stop Solution | 25 mL | 4 C |
