Alkaline Phosphatase Assay Kit

Catalog No. ABIN1000247

Product Details

Target: Alkaline Phosphatase (ALP)

Detection Range: 2-800 U/L

Minimum Detection Limit: 2 U/L

Application: Activity Assay (AcA)

Sample Type: Serum, Plasma

Characteristics: 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.

Components: Assay Buffer: 50 mL, pH 10.5. Mg Acetate: 1.5 mL. 0.2 M pNPP Liquid: 600 µL. 1 M Calibrator: 10 mL Tartrazine.

Material not included: Pipeting devices and accessories (e.g. multi-channel pipettor). Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.

Application Details

Application Notes: 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.

Protocol: 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.

Procedure using 96-well plate:
1. Transfer 200 µL distilled water (H2O) and 200 µL Calibrator into separate wells of a clear bottom 96-well plate.
2. Carefully transfer 5 to 50 µL samples into other wells.
3. Pipet 150 to 195 µL Working Solution to sample wells. The final reaction volume in the sample wells should be 200 µL. Tap plate briefly to mix.
4. Read OD405nm (t = 0), and again after 4 min (t = 4 min) on a plate reader.

Procedure using Cuvette:
1. Transfer 50 µL samples into 1-cm cuvettes.
2. Pipet 950 µL Working Solution to samples. Mix briefly.
3. Read OD405nm shortly after the mixing, and again after 4 min.

Reagent Preparation:

Equilibrate reagents to room temperature. The Working Solution is prepared 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.

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 : 10 4 cells are washed with PBS and lysed in 0.5 mL 0.2% Triton X-100 in distilled water by shaking for 20 min at room temperature.

Restrictions: For Research Use only

Handling

Storage: 4 °C

References

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Target Details

Target: Alkaline Phosphatase (ALP)

Alternative Name: Alkaline Phosphatase (ALP Products)

Synonyms: ALP Kit, ALP1 Kit, CRASH Kit, ECK0378 Kit, JW0374 Kit, psiA Kit, 2410004D18Rik Kit, AU040643 Kit, AW060375 Kit, ILC Kit, CTACK Kit, ESkine Kit, Akp2 Kit, alp Kit, zgc:56672 Kit, DDBDRAFT_0205491 Kit, DDBDRAFT_0231570 Kit, DDB_0205491 Kit, DDB_0231570 Kit, Actn2lp Kit, Alp Kit, asparaginase like 1 Kit, bacterial alkaline phosphatase Kit, C-C motif chemokine ligand 27 Kit, alkaline phosphatase, liver/bone/kidney Kit, alkaline phosphatase Kit, PDZ and LIM domain 3 Kit, ASRGL1 Kit, phoA Kit, Asrgl1 Kit, CCL27 Kit, alpl Kit, alp Kit, Pdlim3 Kit

Background: Quantitative determination of alkaline phosphatase ALP activity using stable p-nitrophenol phosphate substrate at 405nm.

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. The 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. p-Nitrophenyl phosphate p-nitrophenol + phosphate