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.
- 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)
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
↓ Horse Radish Peroxidase + Detection Reagent
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.
|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)|
|Part # 7023||25X Substrate||-20C|
|Part # 4025||Detection Reagent||-20C|
|Part # 6026||Enzyme A||-20C|
|Part # 6027||Enzyme B||-20C|
|Part # 3060||Phosphate Standard||2-8C|
|Part # 6028||Horseradish Peroxidase Enzyme||2-8C|
|Part # 3059||1X Reaction Buffer||2-8C|
|Part # 3061||Sample Diluent||2-8C|
|1 Black Clear-Bottom 96-Well Plate for Fluorescent Plate Reader|