Amplite® Universal Fluorimetric Kinase Assay Kit *Red Fluorescence*
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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 | 12352200 |
Overview | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Run kinase reaction (20 µL)
- Add ADP Sensor Buffer (20 µL)
- Add ADP Sensor working solution (10 µL)
- Incubate at room temperature for 15 minutes - 1 hour
- Monitor fluorescence Intensity at Ex/Em = 540/590 nm (Cutoff = 570nm)
Important notes
Thaw all the six components at room temperature before use. Black plates are strongly recommended to achieve the best results. The ADP assay should be run at pH from 6.5 to 7.4. Avoid direct exposure of ADP Sensor I (Component B1) to light. Avoid potential ADP contamination from exogenous biological sources.
PREPARATION OF STOCK SOLUTION
1. ADP Sensor I stock solution (50X):
Add 50 µL DMSO (Component B3) into vial of ADP Sensor 1 (Component B1) to make 50X ADP Sensor I stock solution.
2. ADP standard solution (300 mM):
Add 100 µL of ddH2O into ADP Standard (Component C) to make a 300 mM ADP standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/31001
Take 300 mM ADP standard solution and dilute 10,000X in kinase reaction buffer to make 30 µM ADP standard solution. Take 30 µM ADP standard solution and perform 1:3 serial dilution in kinase reaction buffer to get serially diluted of ADP standard solution. Note: Make serial dilutions of ADP standard in the kinase reaction buffer by including a sample without ADP for measuring background fluoresence.
PREPARATION OF WORKING SOLUTION
Add 50 µL of 50X ADP Sensor I stock solution into vial of ADP Sensor II (Component B2) to make ADP Sensor working solution. Note: The reconstituted ADP sensor is not stable, make fresh as needed.
SAMPLE EXPERIMENTAL PROTOCOL
Run Kinase reaction (Reagents are not provided for this step):
- Prepare 20 µL (or 10 µL for 384-well plate) of kinase reaction solution/well as desired. The components of kinase reaction should be optimized as needed (e.g., an optimized buffer system might be required for a specific kinase reaction). In most cases, ADP assay buffer (Component D) can also be used to run kinase reaction if you do not have the optimized kinase buffer.
- The Amplite™ Fluorimetric Kinase Assay Kit is used to determine the ADP formation.
Run Amplite™ ADP assay:
- Add 20 µL of ADP Sensor Buffer (Component A) and 10 µL of ADP Sensor working solution into each well filled with the 20 µL kinase reaction solution to make the total ADP assay volume of 50 µL/well. For a 384-well plate, add 10 µL of ADP Sensor Buffer (Component A) and 5 µL of ADP Sensor into each well filled with the 10 µL kinase reaction solution to make the total ADP assay volume of 25 µL/well.
- Incubate the reaction mixture at room temperature for 15 minutes to 1 hour.
- Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
Generate an ADP calibration curve (Not required for the screening of kinase inhibitors):
- Add 20 µL (for a 96-well plate) or 10 µL (for a 384-well plate)/well of ADP Sensor Buffer (Component A) and 10 µL (for a 96-well plate) or 5 µL (for a 384-well plate) of ADP Sensor into each well of serially diluted ADP standards to make the total volume of 50 µL (for a 96-well plate) or 25 µL (for a 384-well plate) for each reaction.
- Incubate the reaction mixture at room temperature for 15 minutes to 1 hour.
- Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
- Generate an ADP standard curve.
Images




Citations
Authors: Huang, Yuanyuan and Lu, Jianlin and Zhan, Li and Wang, Ming and Shi, Ronghua and Yuan, Xiao and Gao, Xinjiao and Liu, Xing and Zang, Jianye and Liu, Wei and others,
Journal: Journal of Biological Chemistry (2021)
Authors: Xu, Leilei and Ali, Mahboob and Duan, Wenxiu and Yuan, Xiao and Garba, Fatima and Mullen, McKay and Sun, Binwen and Poser, Ina and Duan, Hequan and Lu, Jianlin and others,
Journal: Cell Reports (2021): 109343
Authors: Yu, Ruoying and Wu, Huihui and Ismail, Hazrat and Du, Shihao and Cao, Jun and Wang, Jianyu and Ward, Tarsha and Yang, Fengrui and Gui, Ping and Ali, Mahboob and others,
Journal: Journal of molecular cell biology (2020): 462--476
Authors: Zhao, Gangyin and Cheng, Yubao and Gui, Ping and Cui, Meiying and Liu, Wei and Wang, Wenwen and Wang, Xueying and Ali, Mahboob and Dou, Zhen and Niu, Liwen and others,
Journal: Journal of Biological Chemistry (2019): 576--592
Authors: Huang, Yuejia and Lin, Lin and Liu, Xing and Ye, Sheng and Yao, Phil Y and Wang, Wenwen and Yang, Fengrui and Gao, Xinjiao and Li, Junying and Zhang, Yin and others,
Journal: Cell research (2019): 562--578
Authors: Yu, Ruoying and Wu, Huihui and Ismail, Hazrat and Du, Shihao and Cao, Jun and Wang, Jianyu and Ward, Tarsha and Yang, Fengrui and Gui, Ping and Ali, Mahboob and others, undefined
Journal: Journal of Molecular Cell Biology (2019)
Authors: Lang, Lei and Hou, Yixuan and Chen, Yanlin and Tu, Gang and Tao, Jing and Yang, Dan and Xi, Lei and Fu, Lixin and Sun, Kexin and Yin, Jiali and others, undefined
Journal: Cellular Physiology and Biochemistry (2018): 2972--2988
Authors: Yun, Taikangxiang and Qin, Tan and Liu, Ying and Lai, Luhua
Journal: ChemMedChem (2016): 713--717
Authors: Jiang, Hao and Wang, Wenwen and Zhang, Yin and Yao, William W and Jiang, Jiying and Qin, Bo and Yao, Wendy Y and Liu, Fusheng and Wu, Huihui and Ward, Tarsha L and others, undefined
Journal: Journal of Biological Chemistry (2015): 28272--28285
Authors: Yan, Maomao and Chu, Lingluo and Qin, Bo and Wang, Zhikai and Liu, Xing and Jin, Changjiang and Zhang, Guanglan and Gomez, Marta and Hergovich, Alex and er , undefined and Chen, Zhengjun and others, undefined
Journal: Scientific reports (2015): 10449
References
Authors: Li Y, Xie W, Fang G.
Journal: Anal Bioanal Chem (2008): 2049
Authors: Shults MD, Kozlov IA, Nelson N, Kermani BG, Melnyk PC, Shevchenko V, Srinivasan A, Musmacker J, Hachmann JP, Barker DL, Lebl M, Zhao C.
Journal: Chembiochem (2007): 933
Authors: Rykx A, Vancauwenbergh S, De Kimpe L, Janssens K, V and oninck S, Waelkens E, V and enheede JR, Van Lint J.
Journal: Assay Drug Dev Technol (2007): 637
Authors: Vogt A, Lazo JS.
Journal: Methods (2007): 268
Authors: Takeda H, Fukumoto A, Miura A, Goshima N, Nomura N.
Journal: Anal Biochem (2006): 262
Authors: Saldanha SA, Kaler G, Cottam HB, Abagyan R, Taylor SS.
Journal: Anal Chem (2006): 8265
Authors: Trask OJ, Jr., Baker A, Williams RG, Nickischer D, K and asamy R, Laethem C, Johnston PA.
Journal: Methods Enzymol (2006): 419
Authors: Kishimoto A, Ogura T, Esumi H.
Journal: Mol Biotechnol (2006): 17
Authors: Shults MD, Janes KA, Lauffenburger DA, Imperiali B.
Journal: Nat Methods (2005): 277
Authors: Sun H, Low KE, Woo S, Noble RL, Graham RJ, Connaughton SS, Gee MA, Lee LG.
Journal: Anal Chem (2005): 2043
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