Phosphate Assay Kit

Catalog No. ABIN1000335

Product Details

Target: Phosphate

Detection Range: 0.4-50 μM

Minimum Detection Limit: 0.4 μM

Sensitivity: 20 pmol

Characteristics: Reagent 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 µM and 50 µM phosphate.
Fast and convenient: single reagent mix-and-measure 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 to 0.9 are observed in 96- well plates. Can be readily automated on HTS liquid handling systems.

Application Details

Application Notes: 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.

Comment:

Incubation time: The chromogenic reaction is completed within 30 min. Precipitation may occur at high concentrations of phosphate (>100 μM), or in the presence of high concentrations of e.g. proteins and metals. If precipitation occurs, dilute samples in distilled water and repeat the assay. Enzyme reaction buffer. Because any exogenous free phosphate would interfere with the assay, it is important to ensure that the protein preparation, the reaction buffer and lab wares employed in the assay should not contain free phosphate. This can be conveniently checked by adding the Reagent to the buffer and measuring the color formation.

Protocol: Procedure using 96-well plate: Important: The reagent must be brought to room temperature and well shaken before use. The reagent is highly sensitive to phosphate. It is important that all enzyme preparations and assay buffers not contain free phosphate. Lab detergents may contain high levels of phosphate. Make sure that lab wares are washed thoroughly with distilled water and free from contaminating phosphate.
Dilution of phosphate standards. Prepare a 1000 µL 40 µMphosphate Premix solution by mixing 40 µL 1 mM phosphate standard with 960 µL distilled water. Number the tubes. Prepare concentration standards by diluting the Premix.

Restrictions: For Research Use only

Handling

Storage: 4 °C

References

Polewski, Johnson, Foster, Millán, Terkeltaub: "Inorganic pyrophosphatase induces type I collagen in osteoblasts." in: Bone, Vol. 46, Issue 1, pp. 81-90, (2010) (PubMed).

Oborna, Wojewodka, De Sanctis, Fingerova, Svobodova, Brezinova, Hajduch, Novotny, Radova, Radzioch: "Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples." in: Human reproduction (Oxford, England), Vol. 25, Issue 2, pp. 308-16, (2010) (PubMed).

López-Vales, Redensek, Skinner, Rathore, Ghasemlou, Wojewodka, DeSanctis, Radzioch, David: "Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 30, Issue 9, pp. 3220-6, (2010) (PubMed).

Cramp, Kayes, Meyer, Franklin: "Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata." in: The Journal of experimental biology, Vol. 212, Issue Pt 22, pp. 3656-63, (2009) (PubMed).

Huxtable, Zwicker, Poon, Pagliardini, Vrouwe, Greer, Funk: "Tripartite purinergic modulation of central respiratory networks during perinatal development: the influence of ATP, ectonucleotidases, and ATP metabolites." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 29, Issue 47, pp. 14713-25, (2009) (PubMed).

Kang, Shyr, Huang, Tsai, Orimo, Lin, Chang, Huang: "Altered TNSALP expression and phosphate regulation contribute to reduced mineralization in mice lacking androgen receptor." in: Molecular and cellular biology, Vol. 28, Issue 24, pp. 7354-67, (2008) (PubMed).

Chen, Morris, Lawrence, Quinn: "The isolation and structure of membrane lipid rafts from rat brain." in: Biochimie, Vol. 89, Issue 2, pp. 192-6, (2007) (PubMed).

Hough, Polewski, Johnson, Cheeseman, Nolan, Vizor, Rastan, Boyde, Pritzker, Hunter, Fisher, Terkeltaub, Brown: "Novel mouse model of autosomal semidominant adult hypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2." in: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 22, Issue 9, pp. 1397-407, (2007) (PubMed).

Lau, Wu, Sheng, Amoui, Suhr, Baylink: "An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: a mediator of osteoclast activity." in: Journal of cellular biochemistry, Vol. 97, Issue 5, pp. 940-55, (2006) (PubMed).

Target Details

Target: Phosphate

Background: High-throµghput phosphate assay using malachite green method at 620nm.

The Phosphate Assay Kit is based on a proprietary formulation of the malachite green dye. The 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.