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SunRed™ Phosphate

Alkaline phosphatase dose response was measured with the Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Alkaline phosphatase dose response was measured with the Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Ordering information
Price ()
Catalog Number11629
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight422.20
SolventWater
Spectral properties
Excitation (nm)653
Emission (nm)661
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


Molecular weight
422.20
Excitation (nm)
653
Emission (nm)
661
Phosphatase-catalyzed hydrolysis of Sun Red phosphate (SRP) yields the Sun Red fluorophore that can be excited with the 633 nm laser with emission of ~660 nm. Although Sun Red is readily excited at 633 nm with red emission of ~660 nm, SRP has very minimal absorption at 633 nm without red emission, making SRP one of the most sensitive NIR phosphatase sensors. Please do not use DMSO to make stock solution since it significantly increases assay background.

Platform


Fluorescence microplate reader

Excitation620 nm
Emission660 nm
Cutoff640 nm
Recommended plateSolid black

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare and add 10 - 50 µM SunRed™ Phosphate in Tris buffer (50 µL)
  2. Add alkaline phosphatase standards and/or test samples (50 µL)
  3. Incubate at room temperature or 37°C for 30 to 120 minutes
  4. Monitor fluorescence intensity at Ex/Em = 620/660 nm (cut off 640 nm)

Important notes
The following is the recommended protocol for alkaline 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. SunRed™ Phosphate stock solution:
Prepare a 2 to 10 mM stock solution of SunRed™ Phosphate in sterile water. Note: The stock solution should be used promptly. Note: Do not use DMSO, ETOH or METH to make stock solution since it significantly increases assay background. Note: Protect from light.

PREPARATION OF WORKING SOLUTION

SunRed™ Phosphate working solution (2X):
On the day of the experiment, either dissolve SunRed™ Phosphate solid in sterile H2O or thaw an aliquot of the SunRed™ Phosphate 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. SunRed™ Phosphate final concentration of 5 to 25 μM is recommended for measuring alkaline phosphatase activity in solution.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 50 µL of 2X SunRed™ Phosphate working solution into each well of the 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 sample and 25 µL of 2X SunRed™ 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 Ex/Em = 620/660 nm (cut off at 640 nm) with a fluorescence plate reader.

Calculators


Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of SunRed™ Phosphate 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 mM236.855 µL1.184 mL2.369 mL11.843 mL23.685 mL
5 mM47.371 µL236.855 µL473.709 µL2.369 mL4.737 mL
10 mM23.685 µL118.427 µL236.855 µL1.184 mL2.369 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

Excitation (nm)653
Emission (nm)661

Product family


NameExcitation (nm)Emission (nm)Correction Factor (280 nm)
SunRed™ SE5926090.366
SunRed™ Acetate653661-

Citations


View all 1 citations: Citation Explorer
Entwicklung fluoreszenzbasierter Immunoassays f{\"u}r den neuen Inflammationsmarker Chitinase 3-like1 (YKL-40)
Authors: Schmalenberg, Michael Reiner
Journal: (2017)

References


View all 6 references: Citation Explorer
DNA condensation induced by cationic surfactant: a viscosimetry and dynamic light scattering study
Authors: Marchetti S, Onori G, Cametti C.
Journal: J Phys Chem B (2005): 3676
Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates
Authors: Martin K, Hart C, Liu J, Leung WY, Patton WF.
Journal: Proteomics (2003): 1215
Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes
Authors: Top KP, Hatleberg G, Berggren KN, Ryan D, Kemper C, Haugl and RP, Patton WF.
Journal: Electrophoresis (2001): 896
Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP)
Authors: Leira F, Vieites JM, Vieytes MR, Botana LM.
Journal: Toxicon (2000): 1833
A column centrifugation method for the reconstitution in liposomes of the mitochondrial F0F1 ATP synthase/ATPase
Authors: Vazquez-Contreras E, de Gomez-Puyou MT, Dreyfus G.
Journal: Protein Expr Purif (1996): 55
The electrochemical-proton-gradient-activated states of F0F1 ATPase in plant mitochondria as revealed by detergents
Authors: Valerio M, Diolez P, Haraux F.
Journal: Eur J Biochem (1993): 565