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Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*

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 FDP, a sensitive fluorogenic phosphatase substrate, to quantify alkaline phosphatase activity in solutions, in cell extracts as well as on solid surfaces (such as PVDF membranes). The kit provides all the essential components with our optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments.

Example protocol

AT A GLANCE

Protocol Summary
  1. Prepare Alkaline Phosphatase standards or test samples (50 µL)
  2. Add Alkaline Phosphatase working solution (50 µL)
  3. Incubate at RT or 37°C for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutfoff =515 nm)

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

CELL PREPARATION

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

PREPARATION OF STOCK SOLUTIONS

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

FDP stock solution (250X)

Add 100 µL of DMSO (Component D) into the vial of FDP (Component A) to make 250X FDF stock solution. The FDP stock solution should be used promptly.

Alkaline Phosphatase standard solution (100 U/mL)

Add 100 µL of distilled H2O with 0.1% BSA (H2O - 0.1% BSA) into Alkaline Phosphatase Standard (Component C, 10 units) to generate 100 units/mL Alkaline Phosphatase standard solution.

Note        The Alkaline Phosphatase standard solution is not stable.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11953

Alkaline Phosphatase standard
Add 10 µL of 100 units/mL Alkaline Phosphatase standard solution into 990 µL of H2O - 0.1% BSA to generate a 1,000 mU/mL Alkaline Phosphatase standard solution. Take 1,000 mU/mL Alkaline Phosphatase standard solution and perform 1:10 (AS7) and then 1:3 serial dilutions in H2O - 0.1% BSA to get serial dilutions of Alkaline Phosphatase standard (AS6 - AS1).

PREPARATION OF WORKING SOLUTION

Add 20 μL of 250X FDP stock solution into 5 mL of Assay Buffer (Component B) and mix well to prepare Alkaline Phosphatase working solution.

Note        Keep from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Alkaline Phosphatase Standards and test samples in a solid black 96-well microplate. AS = Alkaline Phosphatase Standards (AS1 - AS7, 0.1 to 100 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 (0.1 to 100 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.

    Note        Prepare the cell or tissue samples as desired. Unused serial dilutions of Alkaline Phosphatase standard should be discarded.

  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 at the desired temperature for 10 to 30 minutes, protected from light. Optional: Add 50 µL/well (for a 96-well plate) or 25 µL/well (for a 384-well plate) of Stop Solution (Component E) at the end of 30 minutes incubation.
  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 525 ± 10 nm (Cutoff =515 nm).
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 FDP.

  3. Make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H2O.
  4. Add 100 µL (for a 96-well plate) or 50 µL (for a 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. Optional: add 50 µL/well (for a 96-well plate) or 25 µL/well (for a 384-well plate) of Stop Solution (Component E) at the end of 30 minutes incubation.
  6. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 525 ± 10 nm (Cutoff = 515 nm).

Spectrum

Citations

View all 12 citations: Citation Explorer
Bacteriophage-Based Rapid Bacteria Detection
Authors: Wisuthiphaet, Nicharee
Journal: (2022)
Development and validation of bioengineered intestinal tubules for translational research aimed at safety and efficacy testing of drugs and nutrients
Authors: Jochems, Paulus GM and van Bergenhenegouwen, Jeroen and van Genderen, Anne Metje and Eis, Sophie T and Versprille, Livia JF Wilod and Wichers, Harry J and Jeurink, Prescilla V and Garssen, Johan and Masereeuw, Rosalinde
Journal: Toxicology in Vitro (2019)
Rapid detection of Escherichia coli in beverages using genetically engineered bacteriophage T7
Authors: Wisuthiphaet, Nicharee and Yang, Xu and Young, Glenn M and Nitin, Nitin
Journal: AMB Express (2019): 55
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)

References

View all 109 references: Citation Explorer
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
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
8-Quinolyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase
Authors: Zhu X, Jiang C.
Journal: Clin Chim Acta. (2006)
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 1, 2024

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Spectral properties

Absorbance (nm)

487

Correction Factor (260 nm)

0.32

Correction Factor (280 nm)

0.35

Extinction coefficient (cm -1 M -1)

800001

Excitation (nm)

498

Emission (nm)

517

Quantum yield

0.79001, 0.952

Storage, safety and handling

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

Platform

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components

Alkaline phosphatase dose response was measured with the Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Alkaline phosphatase dose response was measured with the Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Alkaline phosphatase dose response was measured with the Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).