Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared Fluorescence*

Additional fluorescent color(s): 
Image Viewer
Analyze with Quest Graph™Plan a serial dilution
Close (X)
3.0e+32.4e+31.8e+31.2e+3600100101- Dose-responseData legend Generated with Quest Graph™ ALP (mU/mL) RFU Hover mouse to interact
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).
Loading...
 
Unit Size: Cat No: Price (USD): Qty:
500 Tests 11954 $195


Export item/cart as Excel file

Send item/cart as email
EXPORT TO EXCEL X

Export:
EXPORT TO EMAIL X
Important: We request your email address to ensure that the recipient(s) knows you intended for them to see the email, and that it is not junk mail.
Export:
Your Name*:
Your Email*:
Recipient Email*:
Your Personal Message:
Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)646/660
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Enzyme Detection
Phosphatases
Related
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 flurogenic phosphatase substrate, to quantify alkaline phosphatase activity in solutions, in cell extracts as well as on solid surfaces (such as PVDF membranes). This proprietary flurogenic phosphatase substrate generates a fluorescent product that has strongly red fluorescence 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.




Spectrum Advanced Spectrum Viewer

Sorry, your browser does not support inline SVG. Relative Intensity (%) 100 80 60 40 20 0 Sorry, your browser does not support inline SVG.
Sorry, your browser does not support inline SVG. Sorry, your browser does not support inline SVG.
Move mouse over grid to display wavelength & intensity values.

300
400
500
600
700
800
900
Wavelength (nm)





Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.
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 30 to 120 minutes
  4. Monitor fluorescence intensity at Ex/Em = 620/660 nm (Cutoff = 630 nm)

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

Key parameters
Instrument:Fluorescence microplate reader
Excitation:620 nm
Emission:660 nm
Cutoff:630 nm
Recommended plate:Solid black
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. SunRed™ Substrate stock solution (250X):
Add 100 µL of double sterile H2O into the vial of SunRed™ Substrate (Component A) to make 250X SunRedTM Substrate stock solution. The stock solution should be used promptly.

2. Alkaline Phosphatase standard solution (100 U/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 a 100 units/mL Alkaline Phosphatase standard solution.  Note: The Alkaline Phosphatase standard solution is not stable.

Preparation of standard solution
Alkaline Phophatase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11954

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. Take 1,000 mU/mL Alkaline Phosphatase standard solution and perform 1:3 serial dilutions to get serially diluted Alkaline Phosphatase standards (AS7 - AS1) with  H2O - 0.1% BSA.

Preparation of working solution

For one 96-well plate, add 20 μL of 250X SunRed™ Substrate stock solution to 5 mL of Assay Buffer (Component B) and mix well to prepare Alkaline Phosphatase working solution . Note: Keep from light and prepare fresh reaction mixture 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, 0.3 to 300 mU/mL); BL=Blank Control; TS=Test Samples.

BL BL TS TS
AS1 AS1 ... ...
AS2 AS2 ... ...
AS3 AS3    
AS4 AS4    
AS5 AS5    
AS6 AS6    
AS7 AS7    

Table 2. Reagent composition for each well.

Well Volume Reagent
AS1 - AS7 50 µL Serial Dilution (0.3 to 300 mU/mL)
BL 50 µL H2O - 0.1% BSA
TS 50 µL test 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 at for 30 to 120 minutes at the desired temperature, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 620/660 nm (Cutoff = 630 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 SunRed™ 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 each cell well.

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

  6. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 620/660 nm (Cutoff = 630 nm).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate ALP samples. We recommend using the Online Four Parameter Logistics Calculator which can be found at:

https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator

Figure 1. 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).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

The impact of various scaffold components on vascularized bone constructs
Authors: Ahmad Eweida, Matthias Schulte, Oliver Frisch, Ulrich Kneser, Leila Harhaus
Journal: Journal of Cranio-Maxillofacial Surgery (2017)

A Mineralized High Strength and Tough Hydrogel for Skull Bone Regeneration
Authors: Bing Xu, Pengbin Zheng, Fei Gao, Wei Wang, Hongtao Zhang, Xuran Zhang, Xuequan Feng, Wenguang Liu
Journal: Advanced Functional Materials (2016)

DRG axon elongation and growth cone collapse rate induced by Sema3A are differently dependent on NGF concentration
Authors: Andrius Kaselis, Rimantas Treinys, Ruta Vosyliute, Saulius Satkauskas
Journal: Cellular and molecular neurobiology (2014): 289--296

Acute oral toxicity and kinetic behaviors of inorganic layered nanoparticles
Authors: Jin Yu, Hea-Eun Chung, Soo-Jin Choi
Journal: Journal of Nanomaterials (2013): 12

Effect of Some Antihypertensive Drugs on Alkaline Phosphatase and DNA of Mice
Authors: OY El-Khawaga, A El-Waseef, YO Ellazec, MM El-Naggar, Abd M Alla
Journal: International Journal of Genomics and Proteomics (2013): 60

An engineering understanding of the small intestine
Authors: Monica Rosalia Jaime Fonseca
Journal: (2012)

Cytotoxicity and alkaline phosphatase activity evaluation of endosequence root repair material
Authors: Mahmoud Reza Modareszadeh, Peter M Di Fiore, David A Tipton, Narges Salamat
Journal: Journal of endodontics (2012): 1101--1105

Alginate-loaded liposomes can protect encapsulated alkaline phosphatase functionality when exposed to gastric pH
Authors: Alan M Smith, Monica R Jaime-Fonseca, Liam M Grover, Serafim Bakalis
Journal: Journal of agricultural and food chemistry (2010): 4719--4724

Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase
Authors: Krishna M Sinha, Hideyo Yasuda, Madelene M Coombes, Sharon YR Dent, Benoit De Crombrugghe
Journal: The EMBO journal (2010): 68--79






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Certificate of Analysis