ntroduction:The Malachite Green Phosphate Assay Kit is based on quantification of the green complex formed between Malachite Green, molybdate and free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 - 660 nm) or on a plate reader. This non-radioactive colorimetric assay kit has 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 executed in tubes, cuvettes or multi-well plates. The assays can be conveniently performed in 96- and 384-well plates for high-throughput screening of enzyme inhibitors.
Key Features:The Malachite Green Phosphate Assay Kit has been optimized and formulated to provide a sensitive, convenient and robust quantitation of free phosphate liberated from enzyme reactions and natural sources. Key features of the kit are as follows:1. Reagents very stable: Due to our innovative formulation, no precipitation of reagents occurs. Therefore no filtration of reagent is needed prior to assays, as is often required with other commercial kits.
2. High sensitivity and wide detection range: Detection of as little of 1.6pmol of phosphate and useful range between 0.02uM and 40uM phosphate.
3. Fast and convenient: Homogeneous “mix-and-measure” assay allows quantitation of free phosphate within 20 minutes. Compatible with routine laboratory and HTS formats: assays can be performed in tubes, cuvettes or microplates, on spectrophotometers and plate readers.
4. Robust and amenable to HTS: Z’factors of 0.7 to 0.9 are observed in 96- well and 384-well plates. Can be readily automated on HTS liquid handling systems.
Applications:1. Phosphatase Assays: liberation of phosphate from peptide, protein or small molecule substrate. 2. Lipase Assays: liberation of phosphate from phospholipids 3. Nucleoside Triphosphate Assays: Liberation of phosphate from nucleoside triphosphates (ATP, GTP, TTP, CTP etc). 4. Quantitation of Phosphate in phospholipids, proteins and DNAs, etc. 5. Drug Discovery: High-throughput screen for phosphatase inhibitors.
Kit Storage and Stability:Store all kit components at 4°C. Stable for at least 6 months .For maximum recovery of product, centrifuge the original vial prior to removing the cap.
Procedure Using 96-Well Plate:Reagent Preparation: Each assay requires 20ul Working Reagent. Prepare enough Working Reagent by mixing 100 volumes of Reagent A and 1 volume of Reagent B (e.g. 5ml Reagent A and 50ul Reagent B). Working Reagent is stable for at least 1 day at room temperature.
Important: The reagents must be brought to room temperature before use. Before each assay, it is important to check that all enzyme preparations and assay buffers do not contain free phosphate. This can be conveniently tested by adding 20ul of the Working Reagent to 80ul sample solution. The blank OD values at 620 nm should be lower than 0.2. If the OD readings are higher than 0.2, check water phosphate level. Double distilled water usually have OD readings lower than 0.1. Lab detergents may contain high levels of phosphate. Make sure that lab wares are free from contaminating phosphate after thorough washes.
1. Preparation of phosphate standards. Prepare a Premix solution containing 40uM phosphate by pipetting 40ul 1mM phosphate standard to 960ul distilled water or enzyme reaction buffer. Number the tubes.
Dilute standards as shown in the follow ing Table. Pipette 80ulstandard in duplicate into wells of a clear-bottom 96-well plate. Add blank controls containing water or reaction buffer only.
2. Transfer 80ul test samples into separate wells of the plate
Note: In the case of enzyme reactions, the reaction may be terminated by adding a specific inhibitor, or can be stopped directly by the addition of the Working Reagent. Dilution of reaction mixture maybe necessary prior to the assay (see General Considerations). For ATPase or GTPase assays, the ATP or GTP concentration should be lower than 0.25mM. If the reaction mixture contains > 0.25mM ATP or GTP, dilute samples in distilled water. For example, if the ATPase reaction contains 1mM ATP, at the end of reaction dilute reaction mixture 4-fold in water prior to the assay.
3. Add 20ul of Working Reagent to each well. Mix gently by tapping the plate.
4. Incubate for 30 min at room temperature for color development.
5. Measure absorbance at 600nm - 660nm (620nm) on a plate reader.
Note: For assays in 384-well plates, the procedures are the same, except that the volume of the standard and sample solution should be 40ul and that of the Working Reagent should be 10ul.
Data Analysis:Plot OD620nm versus phosphate standard concentrations. Determine sample phosphate concentrations from the standard curve.
