Fluoro Phosphate

Product Description

Introduction

Phosphorus is essential for multiple and diverse biological functions, including cellular signal transduction, mineral meatbolism, and energy exchange. Elevated levels of serum phosphate may be associated with a higher risk of death and adverse cardiovascular outcomes in people with prior myocardial infarction, and is the subject of large amount of research.

Key Benefits

• Quantitation of phosphate anion in blood, plasma and serum samples.
• Sensitive – can detect upto 200nM phosphate
• Easy to use – Fluorescence (ex/em 535/585nm or Absorbance readout @ 570nm)

Assay Principle

Cell Technology’s Phosphate detection kit provides a simple, one-step fluorimetric or colorimetric method for determination of phosphate in serum and plasma samples. The assay is based on an enzyme-coupled reaction that detects inorganic phosphate. The substrate is converted to Hydrogen peroxide in presence of inorganic phosphate in a coupled enzymatic reaction. The Hydrogen Peroxide then reacts with the detection reagent in a 1:1 stoichiometry in presence of Horse Radish Peroxidase to produce the stable fluorescent product.

Substrate + Pi

↓ Coupled enzyme reaction

H2O2

↓ Horse Radish Peroxidase + Detection Reagent

Fluorescent Product

λmax 570nm. λ ex/em 535/585nm.

Colorimetric assay can be read on a spectrophotometer at 570nm.

Fluorescence is measured at excitation 530nm and emission 585nm.

Fig.1 Phosphate standard curve was generated using fluorimetric detection: excitation 535nm and emission 585nm. Incubation time= 60 minutes at 37oC. Standard curve range 1.5625 µM to 100µM. Graph was plotted using 4PL non-linear regression.

Fig.2 Phosphate standard curve inset: R2 =0.9835. Incubation time=60 minutes at 37oC. Standard curve range 1.5625µM to 25µM. Graph plotted with Linear regression.

References

• Mohanty, J.G. et al : A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative, J.Immunological Methods, 202, issue 2, P.133-141 (1997)
• Tonelli, M. et al : Relation between serum phosphate level and cardiovascular event rate in people with coronary disease, Circulation,112, P.2627-2633 (2005)

Kit contents and Long Term storage