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FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*

Chemical structure for FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*
Chemical structure for FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*
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Catalog Number11600
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Physical properties
Molecular weight560.39
SolventWater
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 yield0.79001, 0.952
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


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

Excitation490 nm
Emission514 nm
Cutoff500 nm
Recommended plateSolid black

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare 10 - 50 µM Phosphate in Tris buffer (50 µL)
  2. Add phosphatase standards and/or test samples
  3. Incubate at room temperature or 37°C for 30 to 120 minutes
  4. 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

  1. 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.

  2. Incubate the reaction for 30 to 120 minutes at the desired temperature, protected from light.

  3. Monitor the fluorescence increase at an appropriate filter set with a fluorescence plate reader.

  4. 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 mg0.5 mg1 mg5 mg10 mg
1 mM178.447 µL892.236 µL1.784 mL8.922 mL17.845 mL
5 mM35.689 µL178.447 µL356.894 µL1.784 mL3.569 mL
10 mM17.845 µL89.224 µL178.447 µL892.236 µL1.784 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

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 yield0.79001, 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
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
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)
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
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)
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)

References


View all 33 references: Citation Explorer
Enzyme-nanoparticle functionalization of three-dimensional protein scaffolds
Authors: Hill RT, Shear JB.
Journal: Anal Chem (2006): 7022
TNP-8N3-ADP photoaffinity labeling of two Na,K-ATPase sequences under separate Na+ plus K+ control
Authors: Ward DG, Taylor M, Lilley KS, Cavieres JD.
Journal: Biochemistry (2006): 3460
Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development
Authors: Devader C, Webb RJ, Thomas GM, Dale L.
Journal: Gene (2006): 135
Immunoassay for B. globigii spores as a model for detecting B. anthracis spores in finished water
Authors: Farrell S, Halsall HB, Heineman WR.
Journal: Analyst (2005): 489
Controlled initiation of enzymatic reactions in micrometer-sized biomimetic compartments
Authors: Karlsson A, Sott K, Markstrom M, Davidson M, Konkoli Z, Orwar O.
Journal: J Phys Chem B Condens Matter Mater Surf Interfaces Biophys (2005): 1609
Protein tyrosine phosphatase: enzymatic assays
Authors: Montalibet J, Skorey KI, Kennedy BP.
Journal: Methods (2005): 2
Assaying Cdc25 phosphatase activity
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
Flow injection analysis in a microfluidic format
Authors: Leach AM, Wheeler AR, Zare RN.
Journal: Anal Chem (2003): 967
Measuring the specific activity of the CD45 protein tyrosine phosphatase
Authors: Rider DA, Young SP.
Journal: J Immunol Methods (2003): 127