Phosphate Assay Kit, BioAssay™, Colorimetric

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.||Key Features:|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 20pmoles of phosphate and useful range between 0.4uM and 50uM 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.||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.||Kit Contents:|Storage conditions. The Reagent and standard is stable for 12 months when stored at 4°C.|Precautions: reagents are for research use only. This reagent contains 0.44 M sulfuric acid. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|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.|1. Dilution of phosphate standards. Prepare a 1000ul 40uM phosphate Premix solution by mixing 40ul 1mM phosphate standard with 960ul distilled water. Number the tubes. Prepare concentration standards by diluting the Premix as shown in the The phosphate concentrations in the tubes are given below.|Catalog # Size (assays) Reagent Standard|XXXX-XXX 500 50ml 1ml 1mM phosphate|XXXX-XXX 10,000 1,000ml customized|Transfer 50ul diluted standard in duplicate to wells in a clear-bottom 96-well plate. Store diluted standard at 4°C for future use.|No Premix+H2O|Final Vol (ul)|Phosphate|Conc (uM)|pmoles Phosphate|in 50ul|1 200ul+0ul 200 40 2,000|2 160ul+40ul 200 32 1,600|3 120ul+80ul 200 24 1,200|4 80ul+120ul 200 16 800|5 60ul+140ul 200 12 600|6 40ul+160ul 200 8 400|7 20ul+180ul 200 4 200|8 0ul+200ul 200 0 0|2. Transfer 50ul test sample (e.g. enzyme reaction) in duplicate into wells of the microplate. In the case of enzyme reactions, the reaction may be terminated by either adding a specific inhibitor, or can be stopped directly by the addition of the Reagent. Reaction buffer can be added as a blank control for the samples.|3. Add 100ul of the Reagent to each well. Mix by tapping the plate.|4. Incubate for 30 min at room temperature for color development.|5. Measure absorbance at 620 nm (600 nm-660nm) on a plate reader.|Procedure using Cuvette:|For cuvette assays, add 800ul Reagent to 400ul sample and standards. Perform the assay as described for the microplate assay.||General Considerations:|Incubation time. The chromogenic reaction is completed within 30 min. Precipitation may occur at high concentrations of phosphate (>100uM), 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.||Data Analysis:|Plot pmoles phosphate versus OD620nm for the standard curve. Use linear regression analysis to determine amount of free phosphate in the test samples.