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Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Blue 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 MUP, a 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 working solution (50 µL)
  2. Add Alkaline Phosphatase standards and/or test samples (50 µL)
  3. Incubate at RT or 37 °C for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 360/450 nm (Cutoff = 435 nm)

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. MUP Plus™ stock solution (250X):
Add 100 µL of sterile H2O into the vial of MUP Plus™ (Component A) to make 250X MUP Plus™ stock solution. The MUP Plus™ stock solution should be used promptly.

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

PREPARATION OF STANDARD SOLUTION

Alkaline Phosphatase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11952

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 standard solution to perform 1:10 in H2O-0.1% BSA to get 100 mU/mL standard solution (AS7). Then take 100 mU/mL 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 portion of diluted Alkaline Phosphatase standard solution should be discarded.

PREPARATION OF WORKING SOLUTION

Add 20 μL of 250X MUP Plus™ stock solution into 5 mL  Assay Buffer (Component B) to make Alkaline Phosphatase working solution. Note: Keep from light. Prepare fresh Alkaline Phosphatase working solution for each experiment.

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 black 96-well microplate. AS=Alkaline Phosphatase Standards (AS1 - AS7, 0.1 - 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 Dilutions (0.1 to 100 mU/mL)
BL50 µLH2O - 0.1% BSA
TS50 µLtest sample


Run alkaline phosphate 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 at the desired temperature for 10 to 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 360 ± 10 nm, Emission = 450 ± 10 nm (Cutoff = 435 nm).

Run alkaline phosphatase assay in cells:

  1. Treat the cells as desired.

  2. Add equal volume of Alkaline Phosphatase working solution into each cell well (such as 100 µL/96-well plate, or 50 µL/384-well plate). Note: Alternatively, remove the growth medium from the cell plate, and make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H2O. Then add 100 µL (for a 96-well plate) or 50 uL (for a 384-well plate) of 1:1 diluted working solution into the cell wells.

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

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 360 ± 10 nm, Emission = 450 ± 10 nm (Cutoff = 435 nm).

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*4985178000010.79001, 0.9520.320.35
Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared Fluorescence*592609---0.366

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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Spectral properties

Excitation (nm)

360

Emission (nm)

448

Storage, safety and handling

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

Platform

Fluorescence microplate reader

Excitation360 nm
Emission450 nm
Cutoff435 nm
Recommended plateSolid black

Components

Alkaline phosphatase dose response was measured with Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.01 mU/well of alkaline phosphatase can be detected with 30 minutes incubation (n=3). RFU read at Ex/Em = 360/450 nm (Cutoff = 435 nm).
Alkaline phosphatase dose response was measured with Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.01 mU/well of alkaline phosphatase can be detected with 30 minutes incubation (n=3). RFU read at Ex/Em = 360/450 nm (Cutoff = 435 nm).
Alkaline phosphatase dose response was measured with Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.01 mU/well of alkaline phosphatase can be detected with 30 minutes incubation (n=3). RFU read at Ex/Em = 360/450 nm (Cutoff = 435 nm).