Description
Alkaline Phosphatase Assay Kit (Colorimetric) (BA0013) (BA0013)
The Alkaline Phosphatase Assay Kit (Colorimetric) (SKU: BA0013) provides a colorimetric kinetic method for measuring alkaline phosphatase (ALP) activity directly in biological samples without pretreatment. ALP catalyses the hydrolysis of phosphate esters in an alkaline environment and is found mainly in liver and bone, with markedly raised serum levels associated with biliary obstruction, cirrhosis and other conditions. The improved method uses p-nitrophenyl phosphate, which is hydrolysed by ALP into a yellow product with maximal absorbance at 405 nm, and the reaction rate is directly proportional to enzyme activity. Requiring only 5 µL of serum or plasma, the assay is linear from 2 to 800 U/L and can be completed within 5 minutes. All reagents are compatible with high-throughput liquid handling instruments.
| Product Name: | Alkaline Phosphatase Assay Kit (Colorimetric) (BA0013) |
| SKU: | BA0013 |
| Detection Method: | Colorimetric kinetic (OD 405 nm) |
| Detection Range: | 2 to 800 U/L (5 µL serum/plasma); detection limit 2 U/L |
| Sample Type: | ['Serum', 'Plasma', 'Cell culture media', 'Cell lysate'] |
| Species Reactivity: | All |
| Assay Time: | Within 5 minutes |
| Kit Size: | 250 Assays |
| Equipment Required: | Microplate reader |
| Storage: | pNPP Liquid at -20°C; all other components at 4°C |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
Alkaline phosphatase (ALP) catalyses the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation of an organic radical and inorganic phosphate. In mammals, this enzyme is found mainly in the liver and bones. A marked increase in serum ALP levels has been associated with malignant biliary obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and sarcoidosis. This kit is designed to measure ALP activity directly in biological samples without pretreatment. The improved method utilises p-nitrophenyl phosphate that is hydrolysed by ALP into a yellow coloured product (maximal absorbance at 405 nm). The rate of the reaction is directly proportional to the enzyme activity.
- High sensitivity and wide linear range. Use 5 µL serum or plasma sample. The detection limit is 2 U/L, linear up to 800 U/L.
- Homogeneous and simple procedure. Simple mix-and-measure procedure allows reliable quantitation of ALP activity within 5 minutes.
- Robust and amenable to HTS. All reagents are compatible with high-throughput liquid handling instruments.
- Direct Assays: ALP activity in serum, plasma and other sources.
- Characterisation and Quality Control for ALP production.
- Drug Discovery: high-throughput screen for ALP inhibitors and evaluation of ALP 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 reagents to room temperature. Prepare the Working Solution by mixing, for each 96-well assay, 200 µL Assay Buffer, 5 µL Mg Acetate (final 5 mM) and 2 µL pNPP liquid substrate (10 mM); fresh reconstitution is recommended, although the Working Solution is stable for at least one day at room temperature. |
| 2 | Prepare samples: ALP is stable for 48 hours at 4°C and 2 months at -20°C, and EDTA, oxalate, fluoride and citrate should be avoided as they inhibit ALP. Serum, plasma (no EDTA/citrate, ideally unhemolysed) and cell culture media can be assayed directly. For intracellular ALP, wash 10,000 cells with PBS and lyse in 0.5 mL 0.2% Triton X-100 in distilled water by shaking for 20 min at room temperature. |
| 3 | Transfer 200 µL distilled water and 200 µL Calibrator into separate wells of a clear-bottom 96-well plate. |
| 4 | Carefully transfer 5 to 50 µL samples into other wells. |
| 5 | Pipette 150 to 195 µL Working Solution to sample wells so that the final reaction volume is 200 µL, then tap the plate briefly to mix. |
| 6 | Read OD405nm at t = 0, and again after 4 min on a plate reader. |
ALP activity (IU/L) = [(ODSample t - ODSample 0) x Reaction Vol] / [(ODCalibrator - ODH2O) x Sample Vol x t] x 35.3, where ODSample t and ODSample 0 are OD405nm values of the sample at time t (e.g. 4 min) and 0 min and t is the incubation time in minutes. For p-nitrophenol the molar absorptivity is 18.75 mM-1 cm-1, and the light path is derived from the Calibrator. If sample ALP activity exceeds 800 IU/L, dilute samples in saline, repeat and multiply by the dilution factor; the incubation can be prolonged for low-activity samples. For a cuvette assay, transfer 50 µL sample plus 950 µL Working Solution and calculate ALP activity as 266.7 x (ODSample 4 - ODSample 0).
| Component | Quantity | Storage |
| Assay Buffer (pH 10.5) | 50 mL | 4°C |
| Mg Acetate (0.2 M) | 1.5 mL | 4°C |
| pNPP Liquid (1 M) | 600 µL | -20°C |
| Calibrator (Tartrazine) | 10 mL | 4°C |
