General descriptionPhosphate is one of the most abundant minerals in the human body and contains about 1 % of total body weight. Phosphate homeostasis is maintained by the several regulators such as parathyroid hormone (PTH), calcitriol and phosphatonins. Maintenance of phosphate homeostasis is essential for various biological processes, such as energy metabolism, cell signaling, regulation of protein synthesis, skeletal development and bone integrity. An adequate level of phosphate is needed for the process of apoptosis of mature chondrocytes in the growth plate. Phosphate deficiency is associated with the bone pathology and clinical illness.
ApplicationSuitable for phosphate determination in enzyme reactions with free phosphate releases.
This kit may be used to measure the liberation of free orthophosphate in the following applications:
- Phosphatase Assays: liberation of phosphate from peptide, protein, or small molecule substrate.
- Lipase Assays: liberation of phosphate from phospholipids
- Nucleoside Triphosphatase Assays: liberation of phosphate from nucleoside triphosphates (ATP, GTP, TTP, CTP etc).
- Quantitation of phosphate in phospholipids, proteins and DNAs, etc.
- Drug Discovery: high-throughput screen for phosphatase inhibitors.
Biochem/physiol ActionsThe Phosphate Assay Kit is based on a proprietary formulation of the malachite green dye. The kit reagent forms a blue colored complex with free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 - 660 nm) or on a plate reader. The non-radioactive colorimetric assay kits have been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be performed in tubes, cuvettes or multi-well plates. The assays can be conveniently executed in 96-well plates for high-throughput screening of enzyme inhibitors.
Features and BenefitsReagent very stable: Due to our innovative formulation, no precipitation of reagent occurs. Therefore no filtration of reagent is needed prior to assays, as is often required with other commercial kits.
- High sensitivity and wide detection range: detection of as little of 20 pmoles of phosphate and useful range between 0.4–50 µM phosphate.
- Fast and convenient: homogeneous “mix-andmeasure” assay allows quantitation of free phosphate within 30 minutes.
- Compatible with routine laboratory and HTS formats: assays can be performed in tubes, cuvettes, or microplates, on spectrophotometers and plate readers.
- Robust and amenable to HTS: Z factors of 0.7–0.9 are observed in 96 well plates. Can be readily automated on HTS liquid handling systems.
