SunRed™ Phosphate

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2.8e+32.1e+31.4e+37001010.1- Dose-ResponseData legend Generated with Quest Graph™ ALP (mU/mL) RFU Hover mouse to interact
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).
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5 mg 11629 $195


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)652/660
MW422.20
SolventWater
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Phosphatases
Related Protein Phosphatase
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.




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.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Wavelength (nm)





Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
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.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:620 nm
Emission:660 nm
Cutoff:640 nm
Recommended plate:Solid black
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.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate ALP samples. We recommend using the Online Four Parameter Logistics Calculator which can be found at:

https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator

Figure 1. 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).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References

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, Haugland 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






Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Certificate of Analysis