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How to Use Feyond-A300 for Quantitative Detection of Pyrophosphate

Popularity:288 Published Date:2022-04-11 14:35

Introduction

Pyrophosphate is widely present inside and outside the cell. The pyrophosphate in the cell mainly comes from mitochondria, which cannot pass through the cell membrane by free diffusion, and needs to be transferred to the outside of the cell through the progressive tonic protein channel on the cell membrane. Part of the extracellular pyrophosphate comes from the transmembrane transport of intracellular pyrophosphate, and the other part is generated by a series of extracellular enzymes located on the cell membrane.

Pyrophosphate, a natural substrate for alkaline phosphatase, is produced by many chemical reactions, such as hydrolysis of citrate and ATP, DNA replication and RNA polymerization, cyclic adenosine monophosphate (c-AMP) formation, and formation of fatty acid coenzyme A esters. Fluorescence-based pyrophosphate detection is simpler, more reliable, and more sensitive.

 

Material

• Pyrophosphate assay kit (Merck: MAK168)

• 96-well black opaque plate (Thermo Fisher: 237105)

• Feyond-A300 multi-mode microplate reader

 

Method

• Standard Preparation

Primary liquid concentration

Primary liquid volume μL

Diluent volume μL

Final standard concentration

50mM

10

490

1mM

1mM

50

450

100uM

100uM

45

105

30uM

100uM

15

135

10uM

100uM

4.5

145.5

3uM

10uM

15

135

1uM

3uM

15

135

0.3uM

1uM

15

135

0.1uM


• 
Operation Steps

Prepare the Master Reaction Mix working solution according to the corresponding product instructions. Add 50ul of working solution to each well of a 96-well black flat-bottom microplate, and then add 50ul of pyrophosphate standard to each well, and set 3 duplicate wells for each standard. After mixing, incubate in the dark for 15 minutes, and start the instrument detection.

• Instrument Parameter Setting

Reagent wavelength

EX/EM: 316/456

PMT gain

Middle and high

Stabilization time

150ms

Integration time

40us

 

Figure 1. Standard layout and parameter settings

 

Result

The built-in data processing software of Feyond-A300 can directly fit the standard curve between the concentration of pyrophosphate standard and the fluorescence intensity. Using the four-parameter (4PL) fitting method, the curve equation and curve fitting degree R² are given. The standard curve performs high-precision determination of samples without computer operation, and the report includes information such as standard curve and sample concentration.

 

Figure 2. Pyrophosphate standard curve

 

Summary

As a natural substrate, pyrophosphate plays an important role in a variety of physiological processes and is also closely related to a variety of diseases. Allsheng Feyond-A300 multi-mode microplate reader and high-precision kit can achieve accurate quantification of pyrophosphate.



 

 

Reference

[1] Liu Mengyuan, Wu Liling, Wang Jinyu Pyrophosphate and vascular calcification [J] Advances in Physiological Sciences, 2016, 47 (6): 5

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