Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
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 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | SDSProtocol |
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 |
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 515 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
- Prepare Alkaline Phosphatase standards or test samples (50 µL)
- Add Alkaline Phosphatase working solution (50 µL)
- Incubate at RT or 37°C for 10 - 30 minutes
- 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
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.
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
https://www.aatbio.com/tools/serial-dilution/11953
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.
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 (0.1 to 100 mU/mL) |
BL | 50 µL | H2O - 0.1% BSA |
TS | 50 µL | test sample |
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.
- 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.
- 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.
- Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 525 ± 10 nm (Cutoff =515 nm).
- Treat the cells as desired.
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.
- Make 1:1 dilution of the 5 mL Alkaline Phosphatase working solution with 5 mL distilled H2O.
- 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.
- 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.
- Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 525 ± 10 nm (Cutoff = 515 nm).
Spectrum
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 |
Product Family
Name | Excitation (nm) | Emission (nm) | Correction Factor (280 nm) |
Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Blue Fluorescence* | 360 | 448 | - |
Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared Fluorescence* | 592 | 609 | 0.366 |
Images
Citations
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)
Authors: Wisuthiphaet, Nicharee and Yang, Xu and Young, Glenn M and Nitin, Nitin
Journal: AMB Express (2019): 55
Authors: Eweida, Ahmad and Schulte, Matthias and Frisch, Oliver and Kneser, Ulrich and Harhaus, Leila
Journal: Journal of Cranio-Maxillofacial Surgery (2017)
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)
Authors: Kaselis, Andrius and Treinys, Rimantas and Vosyliute, Ruta and Satkauskas, Saulius
Journal: Cellular and molecular neurobiology (2014): 289--296
Authors: Yu, Jin and Chung, Hea-Eun and Choi, Soo-Jin
Journal: Journal of Nanomaterials (2013): 12
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
Authors: Fonseca, Monica Rosalia Jaime
Journal: (2012)
Authors: Modareszadeh, Mahmoud Reza and Di Fiore, Peter M and Tipton, David A and Salamat, Narges
Journal: Journal of endodontics (2012): 1101--1105
References
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Journal: Ann Clin Biochem (2006): 207
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Journal: Am J Physiol Endocrinol Metab (2004): E648
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Journal: J Biochem (Tokyo) (2003): 63
Authors: Chen YX, Huang AL, Qi ZY, Shan YL, Sun H.
Journal: Hepatobiliary Pancreat Dis Int (2003): 553
Authors: Champion EE, Glazier JD, Greenwood SL, Mann SJ, Rawlings JM, Sibley CP, Jones CJ.
Journal: Placenta (2003): 453
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Journal: Proteomics (2003): 1215
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