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Amplite® Luminometric Alkaline Phosphatase Assay Kit *Luminescence*

Alkaline phosphatase is a highly sensitive enzyme for ELISA, immuno-histochemical, Northern, Southern and Western blot applications. It is widely used in various biological assays (in particular, immunoassays) and ELISA-based diagnostics. This Amplite® Alkaline Phosphatase Assay Kit uses a proprietary luminogenic phosphatase substrate, to quantify alkaline phosphatase activity in solutions as well as in cell extracts. This proprietary phosphatase substrate generates a luminescent product that produces strong luminescence upon interaction with phosphatase. The kit provides all the essential components with our optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments. It has extremely high sensitivity, and can be used for the assays that require demanding sensitivity.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare Alkaline Phosphatase working solution (50 µL)
  2. Add Alkaline Phosphatase standards and/or test samples (50 µL)
  3. Incubate at RT for 30 - 60 minutes
  4. Add Assay Buffer (Component D) (50 µL)
  5. Incubate at RT for 10 - 30 minutes
  6. Monitor luminescence intensity

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Alkaline Phosphatase standard solution (100 U/mL):
Add 100 µL of distilled H2O with 0.1% BSA (H2O - 0.1% BSA) into the vial of Alkaline Phosphatase Standard (Component C, 10 units) to generate a 100 units/mL Alkaline Phosphatase standard solution. Note: The Alkaline Phosphatase standard solution is not stable.

PREPARATION OF STANDARD SOLUTION

Alkaline Phosphatase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11956

Add 10 µL of 100 units/mL Alkaline Phosphatase standard solution into 990 µL of H2O - 0.1% BSA to generate 1,000 mU/mL Alkaline Phosphatase standard solution. Take 1,000 mU/mL Alkaline Phosphatase standard solution and perform 1:100 in H2O - 0.1% BSA to get 10 mU/mL Alkaline Phosphatase standard solution (AS7). Then take 10 mU/mL Alkaline Phosphatase standard solution (AS7) and perform 1:3 serial dilutions in H2O - 0.1% BSA to get serially diluted Alkaline Phosphatase standards (AS6 - AS1). Note: Unused serial dilutions of Alkaline Phosphatase standard should be discarded. 

PREPARATION OF WORKING SOLUTION

Mix the whole content of Phosphatase Substrate (Component A) with Reaction Buffer (Component B) to make Alkaline Phosphatase working solution. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Alkaline Phosphatase standards and test samples in a solid white 96-well microplate. AS=Alkaline Phosphatase Standards (AS1 - AS7, 0.01 to 10 mU/mL); BL=Blank Control; TS=Test Samples. 

BLBLTSTS
AS1AS1......
AS2AS2......
AS3AS3  
AS4AS4  
AS5AS5  
AS6AS6  
AS7AS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
AS1 - AS750 µLSerial Dilution (0.01 to 10 mU/mL)
BL50 µLH2O - 0.1% BSA
TS50 µLtest sample

Run alkaline phosphatase assay in supernatants:

  1. Prepare Alkaline Phosphatase standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of Alkaline Phosphatase working solution to each well of Alkaline Phosphatase standard, blank control, and test samples to make the total Alkaline Phosphatase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Alkaline Phosphatase working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.

  4. Add 50 µL of Assay Buffer (Component D) into each well of Alkaline Phosphatase standard, blank control, and test samples with assay reaction mixture to make the total Alkaline Phosphatase assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Assay Buffer (Component D) into each well, for a total volume of 75 µL/well.

  5. Incubate the reaction for 10 to 30 minutes at room temperature, protected from light.

  6. Monitor the luminescence increase with a standard luminescence plate reader.

Run alkaline phosphatase assay in cells:

  1. Treat the cells as desired.

  2. Remove the growth medium completely from the cell plate. Note: It is important to remove the growth medium completely from the cell plate due to the interference of the growth medium with the phosphatase substrate.

  3. Make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H2O.

  4. Add 100 µL (96-well plate) or 50 uL (384-well plate) of 1:1 diluted Alkaline Phosphatase working solution into the cell wells.

  5. Incubate the reaction at the desired temperature for 30 to 60 minutes, protected from light.

  6. Add 50 µL (96-well plate) or 25 uL (384-well plate) of Assay Buffer (Component D) into the cell wells containing working solution.

  7. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.

  8. Monitor the luminescence increase with a standard luminescence plate reader.

Citations

View all 9 citations: Citation Explorer
The impact of various scaffold components on vascularized bone constructs
Authors: Eweida, Ahmad and Schulte, Matthias and Frisch, Oliver and Kneser, Ulrich and Harhaus, Leila
Journal: Journal of Cranio-Maxillofacial Surgery (2017)
A Mineralized High Strength and Tough Hydrogel for Skull Bone Regeneration
Authors: Xu, Bing and Zheng, Pengbin and Gao, Fei and Wang, Wei and Zhang, Hongtao and Zhang, Xuran and Feng, Xuequan and Liu, Wenguang
Journal: Advanced Functional Materials (2016)
DRG axon elongation and growth cone collapse rate induced by Sema3A are differently dependent on NGF concentration
Authors: Kaselis, Andrius and Treinys, Rimantas and Vosyliute, Ruta and Satkauskas, Saulius
Journal: Cellular and molecular neurobiology (2014): 289--296
Acute oral toxicity and kinetic behaviors of inorganic layered nanoparticles
Authors: Yu, Jin and Chung, Hea-Eun and Choi, Soo-Jin
Journal: Journal of Nanomaterials (2013): 12
Effect of Some Antihypertensive Drugs on Alkaline Phosphatase and DNA of Mice
Authors: El-Khawaga, OY and El-Waseef, A and Ellazec, YO and El-Naggar, MM and Alla, Abd M
Journal: International Journal of Genomics and Proteomics (2013): 60

References

View all 109 references: Citation Explorer
8-Quinolyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase
Authors: Zhu X, Jiang C.
Journal: Clin Chim Acta. (2006)
Effects of hydrogen peroxide (H(2)O(2)) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines
Authors: Lee DH, Lim BS, Lee YK, Yang HC.
Journal: Cell Biol Toxicol (2006): 39
The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue
Authors: Ali AT, Penny CB, Paiker JE, Psaras G, Ikram F, Crowther NJ.
Journal: Ann Clin Biochem (2006): 207
Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1
Authors: Ali AT, Penny CB, Paiker JE, van Niekerk C, Smit A, Ferris WF, Crowther NJ.
Journal: Clin Chim Acta (2005): 101
Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study
Authors: Rieu JP, Ronzon F, Place C, Dekkiche F, Cross B, Roux B.
Journal: Acta Biochim Pol (2004): 189
Page updated on December 6, 2024

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Luminescence microplate reader

Recommended plateSolid white

Components

Alkaline phosphatase dose response was measured with Amplite® Luminometric Alkaline Phosphatase Assay Kit in a white 96-well plate using a NovoStar microplate reader (BMG Labtech). As low as 0.001 mU/mL alkaline phosphatase can be detected with 20 minutes incubation (n=3).
Alkaline phosphatase dose response was measured with Amplite® Luminometric Alkaline Phosphatase Assay Kit in a white 96-well plate using a NovoStar microplate reader (BMG Labtech). As low as 0.001 mU/mL alkaline phosphatase can be detected with 20 minutes incubation (n=3).
Alkaline phosphatase dose response was measured with Amplite® Luminometric Alkaline Phosphatase Assay Kit in a white 96-well plate using a NovoStar microplate reader (BMG Labtech). As low as 0.001 mU/mL alkaline phosphatase can be detected with 20 minutes incubation (n=3).