Amplite® Colorimetric Alkaline Phosphatase Assay Kit *Yellow Color*
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Catalog Number | |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | ![]() ![]() |
Platform
Absorbance microplate reader
Absorbance | 400 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare alkaline phosphatase standards and/or test samples (50 µL)
- Add ALP working solution (50 µL)
- Incubate at RT or 37°C for 10 - 30 minutes
- Monitor absorbance increase at 400 nm
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
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
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
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 |
In supernatants:
- 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.
- 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.
- Incubate the reaction at the desired temperature for 10 to 30 minutes, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at 400 nm.
In cells:
-
Treat the cells as desired.
-
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.
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Incubate the reaction at the desired temperature for 30 to 60 minutes, protected from light.
-
Monitor the absorbance increase with an absorbance plate reader at 400 nm.
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 *Green Fluorescence* | 498 | 517 | 0.35 |
Amplite® Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared Fluorescence* | 592 | 609 | 0.366 |
Images
Citations
Authors: Heming, Bo
Journal: (2023)
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
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
Authors: Nandi, Sayantan
Journal: (2022)
Authors: Patel, Dinesh K and Dutta, Sayan Deb and Hexiu, Jin and Ganguly, Keya and Lim, Ki-Taek
Journal: Carbohydrate Polymers (2022): 119077
Authors: Abdelgawad, Latifa Mohamed and Hasan, Mustafa and Sabry, Dina and Abdelgwad, Marwa
Journal: Brazilian Dental Science (2022)
Authors: Diederich, Antje
Journal: (2020)
Authors: Langerak, Nicky and Ahmed, Haysam MM and Li, Yang and Middel, Igor R and Eslami Amirabadi, Hossein and Malda, Jos and Masereeuw, Rosalinde and van Roij, Ren{\'e}
Journal: Frontiers in bioengineering and biotechnology (2020): 763
Authors: Jochems, Paulus GM and Keusters, Willem R and America, Antoine HP and Rietveld, Pascale and Bastiaan-Net, Shanna and Ari{\"e}ns, Renata and Tomassen, Monic MM and Lewis, Fraser and Li, Yang and Westphal, Koen GC and others,
Journal: npj Science of Food (2020): 1--9
Authors: Stein, Merle and Barnea-Zohar, Maayan and Shalev, Moran and Arman, Esther and Brenner, Ori and Winograd-Katz, Sabina and Gerstung, Jennifer and Thalji, Fadi and Kanaan, Moien and Elinav, Hila and others,
Journal: Bone (2020): 115360
References
Authors: Zhu X, Jiang C.
Journal: Clin Chim Acta. (2006)
Authors: Lee DH, Lim BS, Lee YK, Yang HC.
Journal: Cell Biol Toxicol (2006): 39
Authors: Ali AT, Penny CB, Paiker JE, Psaras G, Ikram F, Crowther NJ.
Journal: Ann Clin Biochem (2006): 207
Authors: Ali AT, Penny CB, Paiker JE, van Niekerk C, Smit A, Ferris WF, Crowther NJ.
Journal: Clin Chim Acta (2005): 101
Authors: Rieu JP, Ronzon F, Place C, Dekkiche F, Cross B, Roux B.
Journal: Acta Biochim Pol (2004): 189
Authors: Palermo C, M and uca P, Gazzerro E, Foppiani L, Segat D, Barreca A.
Journal: Am J Physiol Endocrinol Metab (2004): E648
Authors: Chen YX, Huang AL, Qi ZY, Shan YL, Sun H.
Journal: Hepatobiliary Pancreat Dis Int (2003): 553
Authors: Wood SR, Zhao Q, Smith LH, Daniels CK.
Journal: Tissue Cell (2003): 47
Authors: Martin K, Hart C, Liu J, Leung WY, Patton WF.
Journal: Proteomics (2003): 1215
Authors: Champion EE, Glazier JD, Greenwood SL, Mann SJ, Rawlings JM, Sibley CP, Jones CJ.
Journal: Placenta (2003): 453
Application notes
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