Alkaline phosphatase (ALP) catalyzes 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. Marked increase in serum ALP levels, a disease known as hyperalkalinephosphatasemia, has been associated with malignant biliary obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and sarcoidosis.
Simple, direct and automation-ready procedures for measuring ALP activity in serum are becoming popular in Research and Drug Discovery. Alkaline Phosphatase Assay Kit is designed to measure ALP activity directly in biological samples without pretreatment. The improved method utilizes p-nitrophenyl phosphate that is hydrolyzed by ALP into a yellow colored product (maximal absorbance at 405nm). The rate of the reaction is directly proportional to the enzyme activity.
Key Features:High sensitivity and wide linear range. Use 5ul 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 highthroughput liquid handling instruments.
Applications:Direct Assays: ALP activity in serum, plasma and other sources. Characterization and Quality Control for ALP production
Drug Discovery: high-throughput screen for ALP inhibitors and evaluation of ALP inhibitors.
Kit Contents: (250 tests in 96-well plates)Assay Buffer: 50ml, pH 10.5 Mg Acetate: 1.5ml 0.2 M pNPP Liquid: 600ul 1 M Calibrator: 10ml TartrazineStorage conditions. The kit is shipped at room temperature. Store pNPP Liquid at -20°C and other components at 4°C. Shelf life of 12 months after receipt
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.
Procedures:This assay is based on a kinetic reaction. Use of a multi-channel pipettor is recommended. Addition of Working Reagent to samples should be quick and mixing should be brief but thorough. Assays can be executed at room temperature or 37°C
Reagent preparation: equilibrate reagents to room temperature. The Working Solution is prepared by mixing for each 96-well assay, 200ul Assay Buffer, 5ul Mg Acetate (final 5mM) and 2ul pNPP liquid substrate (10mM). Fresh reconstitution is recommended, although the Working Solution is stable for at least one day at room temperature
Sample preparation: ALP is stable for 48 hours at 4°C and 2 months at -20°C. EDTA, oxalate, fluoride, citrate are known inhibitors of ALP and should be avoided in sample preparation. Serum, plasma (no EDTA/citrate, ideally unhemolyzed) and cell culture media can be assayed directly. To measure intracellular ALP, cell lysate can be prepared as follows: 10e4 cells are washed with PBS and lysed in 0.5ml 0.2% Triton X-100 in distilled water by shaking for 20 min at room temperature
Procedure using 96-well plate:1. Transfer 200ul distilled water (H2O) and 200ul Calibrator into separate wells of a clear bottom 96-well plate
2. Carefully transfer 5 to 50ul samples into other wells
3. Pipet 150 to 195ul Working Solution to sample wells. The final reaction volume in the sample wells should be 200ul. Tap plate briefly to mix
4. Read OD405nm (t=0), and again after 4 min (t=4 min) on a plate reader
5. Calculation:: ALP activity of the sample (IU/L=uMol/(L·min)) is=(ODSAMPLE t–ODSAMPLE o) × 1000 × Reaction Volt × e × l × Sample Vol=(ODSAMPLE t–ODSAMPLE o) × Reaction Vol(ODCALIBRATOR–ODH2O) × Sample Vol × tx 35.3ODSAMPLE t and ODSAMPLEo are OD405nm values of sample at time t (e.g. 4)and 0 min. The factor 1000 convertsmMol/L touMol/L. t is the incubationtime (min). For p-nitrophenol, e=18.75mM-1×cm-1. l (light path, cm) is 1 cmfor cuvette, and calculated for 96-well assay from the Calibrator, l =(ODCALIBRATOR-ODH2O)/(e × c).
Procedure using Cuvette:1. Transfer 50ul samples into 1-cm cuvettes
2. Pipet 950ul Working Solution to samples. Mix briefly
3. Read OD405nm shortly after the mixing, and again after 4 min
4. Calculation:: ALP activity of the sample (IU/L) is=(ODSAMPLE t–ODSAMPLE 0) × 1000 × Reaction Volt × e × l × Sample Vol= 266.7 x (ODSAMPLE4–ODSAMPLE0)Note: (1) if sample ALP activity exceeds 800 IU/L, dilute samples insaline and repeat the assay, multiply the result by the dilution factor. (2)incubation can be prolonged for samples with low ALP activity.
Materials Required, But Not Provided:Pipeting devices and accessories (e.g. multi-channel pipettor)
Procedure using 96-well plate:Clear bottom 96-well plates (e.g. Corning Costar) and plate reader
Procedure using cuvette:Spectrophotometer and cuvets for measuring OD 405nm.
Examples:Samples were assayed in duplicate (n=2) using the 96-well plate protocol. The ALP activity (U/L) was 13.4 ± 0.4 for a human serum, 190.4 ± 1.6 for rat serum and 202.8 ± 4.3 for goat serum
Kinetics of ALP reaction in 96-well plate assay with increasing ALP concentration
