FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*
Overview | ![]() ![]() |
See also: Alkaline Phosphatase (ALP), Antibodies and Proteomics, Bioconjugation, Biotin and Streptavidin, Chemical Reagents, Classic Dyes, Rhodamines and Rhodamine Derivatives, Enzyme-Linked Immunosorbent Assay (ELISA), Enzymes, Fluoresceins, Secondary Antibodies
CAS 217305-49-2 | Molecular weight 560.39 | Absorbance (nm) 487 | Correction Factor (260 nm) 0.32 | Correction Factor (280 nm) 0.275 | Extinction coefficient (cm -1 M -1) 800001 | Excitation (nm) 498 | Emission (nm) 517 | Quantum yield 0.79001, 0.952 |
Upon interaction with phosphatases the colorless and non-fluorescent FDP is hydrolyzed to highly fluorescent fluorescein, which exhibits excellent spectral properties that match the optimal detection window of most fluorescence instruments that are equipped with the Argon laser excitation. Alternatively, FDP can also be used to detect phosphatases in a chromogenic mode since the enzymatic product (fluorescein) exhibits a large extinction coefficient (close to 100,000 cm-1mol-1). In some literature, FDP was considered to be one of the most sensitive fluorogenic phosphatase substrates. FDP has been widely used in various ELISA assays. Additionally it is also used to detect tyrosine phosphatases. FDP is thermally unstable, and special cautions need be excised for storing the solid sample and stock solutions.
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 514 nm |
Cutoff | 500 nm |
Recommended plate | Solid black |
Example protocol
AT A GLANCE
Protocol summary
- Prepare 10 - 50 µM Phosphate in Tris buffer (50 µL)
- Add phosphatase standards and/or test samples
- Incubate at room temperature or 37°C for 30 to 120 minutes
- Monitor fluorescence intensity at Ex/Em= 490/514 nm
Important notes
Phosphatase substrates can be detected by phosphatases that may not be specifically listed. The following is the recommended protocol for phosphatase assay in solution. The protocol only provides a guideline, should be modified according to the specific needs.
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. FDP stock solution:
Prepare a 2 to 10 mM stock solution in ddH2O. Note: The stock solution should be used promptly.
PREPARATION OF WORKING SOLUTION
FDP working solution (2X):
On the day of the experiment, either dissolve FDP in ddH2O or thaw an aliquot of the stock solution at room temperature. Prepare a 2X working solution of 10 to 50 µM in 100 mM Tris buffer or buffer of your choice, pH 8 to 9 (not phosphate buffer).
SAMPLE EXPERIMENTAL PROTOCOL
- Add 50 µL of 2X FDP working solution into each well of the phosphatase standard, blank control, and test samples to make the total phosphatase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of sample and 25 µL of 2X Phosphate working solution into each well.
- Incubate the reaction for 30 to 120 minutes at the desired temperature, protected from light.
- Monitor the fluorescence increase at an appropriate filter set with a fluorescence plate reader.
- The fluorescence in blank wells (with the assay buffer only) is used as a control, and is subtracted from the values for those wells with the phosphatase reactions.
Calculators
Common stock solution preparation
Table 1. Volume of Water needed to reconstitute specific mass of FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 178.447 µL | 892.236 µL | 1.784 mL | 8.922 mL | 17.845 mL |
5 mM | 35.689 µL | 178.447 µL | 356.894 µL | 1.784 mL | 3.569 mL |
10 mM | 17.845 µL | 89.224 µL | 178.447 µL | 892.236 µL | 1.784 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
Absorbance (nm) | 487 |
Correction Factor (260 nm) | 0.32 |
Correction Factor (280 nm) | 0.275 |
Extinction coefficient (cm -1 M -1) | 800001 |
Excitation (nm) | 498 |
Emission (nm) | 517 |
Quantum yield | 0.79001, 0.952 |
Citations
View all 6 citations: Citation Explorer
Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state
Authors: Miller, Peter G and Sathappa, Murugappan and Moroco, Jamie A and Jiang, Wei and Qian, Yue and Iqbal, Sumaiya and Guo, Qi and Giacomelli, Andrew O and Shaw, Subrata and Vernier, Camille and others,
Journal: Nature Communications (2022): 1--16
Authors: Miller, Peter G and Sathappa, Murugappan and Moroco, Jamie A and Jiang, Wei and Qian, Yue and Iqbal, Sumaiya and Guo, Qi and Giacomelli, Andrew O and Shaw, Subrata and Vernier, Camille and others,
Journal: Nature Communications (2022): 1--16
Ratiometric fluorescent sensing and imaging of intracellular pH by an AIE-active luminogen with intrinsic phosphatase-like catalytic activity
Authors: Dai, Ling and Mao, Weilin and Hu, Lianzhe and Song, Jiaxing and Zhang, Yan and Huang, Ting and Wang, Min
Journal: Dyes and Pigments (2022): 110436
Authors: Dai, Ling and Mao, Weilin and Hu, Lianzhe and Song, Jiaxing and Zhang, Yan and Huang, Ting and Wang, Min
Journal: Dyes and Pigments (2022): 110436
On-Chip Enrichment System for Digital Bioassay Based on Aqueous Two-Phase System
Authors: Minagawa, Yoshihiro and Nakata, Shoki and Date, Motoki and Ii, Yutaro and Noji, Hiroyuki
Journal: ACS nano (2022)
Authors: Minagawa, Yoshihiro and Nakata, Shoki and Date, Motoki and Ii, Yutaro and Noji, Hiroyuki
Journal: ACS nano (2022)
Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array
Authors: Zhang, Yi and Minagawa, Yoshihiro and Kizoe, Hiroto and Miyazaki, Kentaro and Iino, Ryota and Ueno, Hiroshi and Tabata, Kazuhito V and Shimane, Yasuhiro and Noji, Hiroyuki
Journal: Science advances (2019): eaav8185
Authors: Zhang, Yi and Minagawa, Yoshihiro and Kizoe, Hiroto and Miyazaki, Kentaro and Iino, Ryota and Ueno, Hiroshi and Tabata, Kazuhito V and Shimane, Yasuhiro and Noji, Hiroyuki
Journal: Science advances (2019): eaav8185
Fluorescence quantification of intracellular materials at the single-cell level by an integrated dual-well array microfluidic device
Authors: Wang, Chenyu and Ren, Lufeng and Liu, Wenwen and Wei, Qingquan and Tan, Manqing and Yu, Yude
Journal: Analyst (2019)
Authors: Wang, Chenyu and Ren, Lufeng and Liu, Wenwen and Wei, Qingquan and Tan, Manqing and Yu, Yude
Journal: Analyst (2019)
Multifunctional and Programmable Modulated Interface Reactions on Proteinosomes
Authors: Zhou, Pei and Wu, Shuang and Liu, Xiaoman and Wu, Guangyu and Hegazy, Mohammad and Huang, Xin
Journal: ACS Applied Materials & Interfaces (2018)
Authors: Zhou, Pei and Wu, Shuang and Liu, Xiaoman and Wu, Guangyu and Hegazy, Mohammad and Huang, Xin
Journal: ACS Applied Materials & Interfaces (2018)
References
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Journal: J Phys Chem B Condens Matter Mater Surf Interfaces Biophys (2005): 1609
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Journal: Methods (2005): 2
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Journal: Methods (2005): 2
Assaying Cdc25 phosphatase activity
Authors: Hassepass I, Hoffmann I.
Journal: Methods Mol Biol (2004): 153
Authors: Hassepass I, Hoffmann I.
Journal: Methods Mol Biol (2004): 153
FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli
Authors: Bramkamp M, Gassel M, Altendorf K.
Journal: Biochemistry (2004): 4559
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Journal: Biochemistry (2004): 4559
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Journal: Anal Chem (2003): 967
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Journal: Anal Chem (2003): 967
Measuring the specific activity of the CD45 protein tyrosine phosphatase
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Journal: J Immunol Methods (2003): 127
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