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Amplite® Colorimetric Alkaline Phosphatase Assay Kit *Yellow Color*

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 pNPP, a chromogenic 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 and/or test samples (50 µL)
  2. Add ALP working solution (50 µL)
  3. Incubate at RT or 37°C for 10 - 30 minutes
  4. Monitor absorbance increase at 400 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. pNPP stock solution (100X):
Add 300 µL of distilled H2O into the vial of pNPP (Component A). Mix well. The pNPP stock solution should be used promptly. Note: The solution should be good for 3 - 4 weeks if stored properly.

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

PREPARATION OF STANDARD SOLUTION

Alkaline Phosphate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11950

Add 10 µL of 100 units/mL Alkaline Phosphatase standard solution to 990 µL of H2O - 0.1% BSA to generate a 1,000 mU/mL Alkaline Phosphatase standard solution. Then take 100 µL of 1,000 mU/mL Alkaline Phosphatase standard solution to perform a 1:10 dilution to obtain 100 mU/mL Alkaline Phosphatase standard solution (AS7). Then perform 1:3 serial dilution to obtain remaining standards (AS6 - AS1). Note: The unused portion of diluted alkaline phosphatase standard solution should be discarded.

PREPARATION OF WORKING SOLUTION

Add 50 μL of pNPP Stock solution (100X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.05 mL of pNPP working solution.

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 samples in a white/clear bottom 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


In supernatants:

  1. Prepare alkaline phosphate 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 pNPP working solution to each well of alkaline phosphate standard, blank control, and test samples to make the total alkaline phosphate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of pNPP 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 absorbance increase with an absorbance plate reader at 400 nm.

In cells:

  1. Treat the cells as desired.

  2. Add equal volume of pNPP 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 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 to the cell wells.

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

  4. Monitor the absorbance increase with an absorbance plate reader at 400 nm.

Citations

View all 26 citations: Citation Explorer
Bioengineered intestinal tubules as a tool to test intestinal biological efficacy of lettuce species
Authors: Jochems, Paulus GM and Heming, Bo and Lapin, Dmitry and Moonen, Naomi EL and Van den Ackerveken, Guido and Masereeuw, Rosalinde
Journal: npj Science of Food (2022): 1--8
MDS cells impair osteolineage differentiation of MSCs via extracellular vesicles to suppress normal hematopoiesis
Authors: Hayashi, Yasutaka and Kawabata, Kimihito C and Tanaka, Yosuke and Uehara, Yasufumi and Mabuchi, Yo and Murakami, Koichi and Nishiyama, Akira and Kiryu, Shigeru and Yoshioka, Yusuke and Ota, Yasunori and others,
Journal: Cell Reports (2022): 110805
3D-printable chitosan/silk fibroin/cellulose nanoparticle scaffolds for bone regeneration via M2 macrophage polarization
Authors: Patel, Dinesh K and Dutta, Sayan Deb and Hexiu, Jin and Ganguly, Keya and Lim, Ki-Taek
Journal: Carbohydrate Polymers (2022): 119077

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 November 5, 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

Absorbance microplate reader

Absorbance400 nm
Recommended plateClear bottom

Components

Alkaline phosphatase dose response was measured with the Amplite® Colorimetric Alkaline Phosphatase Assay Kit in a white/clear bottom 96-well plate using a NovoStar microplate reader (BMG Labtech).
Alkaline phosphatase dose response was measured with the Amplite® Colorimetric Alkaline Phosphatase Assay Kit in a white/clear bottom 96-well plate using a NovoStar microplate reader (BMG Labtech).
Alkaline phosphatase dose response was measured with the Amplite® Colorimetric Alkaline Phosphatase Assay Kit in a white/clear bottom 96-well plate using a NovoStar microplate reader (BMG Labtech).