The Malachite Green Phosphate Assay Kit is based on the quantification of the green complex formed between green malachite, molybdate and free orthophosphate. The rapid formation of colour from the reaction can be conveniently measured in a spectrophotometer (600 – 660 nm) or in a plate reader Non-radioactive colourimetric assay kits have been optimized for superior sensitivity and long life. the essay is simple and fast, involving a single phosphate addition step determination. Assays can be run in tubes, cuvettes, or multiwells plates. Assays can be conveniently performed in 96 and 384 wells plates for high throughput screening of enzyme inhibitors.
- Very stable reagent. Due to our innovative formula, there is no precipitation of the reactant is produced. Therefore, filtration of the reagent is not needed before assays, as is often necessary with other commercial kits.
- High sensitivity and wide detection range: detection of as little as 1 mole of phosphate and useful range between 0.02 µM and 40 µM phosphate.
- Fast and convenient: homogeneous “mix and measure” assay allows free phosphate quantification in 20 minutes.
- Compatible with routine laboratories and HTS formats: assays can be carried out in tubes, cuvettes or microplates, in spectrophotometers and plate readers
- Robust and HTS-susceptible: Z’ factors of 0.7 to 0.9 are observed in 96- well and 384-well plates. Can be easily automated in HTS liquid handling systems.
- Phosphatase assays: Phosphate release from peptides, proteins, or small molecule substrates.
- Lipase Assays: Phosphate Release from Phospholipids
- Nucleoside triphosphatase assays: phosphate release from nucleoside triphosphates (ATP, GTP, TTP, CTP, etc.).
- Phosphate quantification in phospholipids, proteins and DNAs, etc.
- Drug Discovery: high throughput screen for phosphatase inhibitors.
Kit Content: 2500 Tests In 96 Well Plate
- Reagent A: 50 ml
- Reagent B: 1 ml
- Standard: 1 ml of 1 mM phosphate
- Storage conditions. The kit is shipped at room temperature. The reagents and standard are stable for one year when stored at 4°C.
- Precautions: Reagents are for research use only. normal precautions for laboratory reagents should be exercised while using the reagents. Please consult the Material Safety Data Sheet for detailed information.
Procedure With 96 Well Plate
- Preparation of reagents. Each assay requires 20 µL of working reagent. Prepare a sufficient working reagent by mixing 100 vol of reagent A and 1. vol. of Reagent B (eg, 5 mL of Reagent A and 50 µL of Reagent B). The working reagent is stable for at least 1 day at room temperature.
Important: The reagent must be at room temperature before use. Before each test, it is important to check that all enzyme preparations and assay buffers do not contain free phosphate. This can be conveniently made by adding 20 µL of working reagent to 80 µL of sample solution. Blank OD values at 620 nm should be less than 0.2. if the OD readings are greater than 0.2, check the phosphate level of the water double distilled water usually has a DO reading less than 0.1. Laboratory detergents can contain high levels of phosphate. Make sure laboratory materials are free of contaminating phosphate after thorough washing.
- Preparation of phosphate standards. Prepare a premix solution containing 40 µM phosphate by pipetting 40 µL of 1 mM phosphate standard to 960 µL of distilled water or enzyme reaction buffer. Number the tubes. Dilute the standards as shown in the following table. 80 µL standard pipet in duplicate in wells of a 96-well clear bottom plate. Add blank controls containing water or reaction buffer only.
- Transfer 80 µL test samples to separate wells of the plate.
Note: In the case of enzymatic reactions, the reaction can be terminated by adding a specific inhibitor, or it can be stopped directly by adding the working reagent. Dilution of the reaction mixture may be necessary before the test (see General considerations). For ATPase or GTPase Assays, the concentration of ATP or GTP should be less than 0.25 mM. Yes, the reaction mix contains > 0.25 mM ATP or GTP, dilute samples in distilled water. For example, if the ATPase reaction contained 1 mM ATP, at the end of the reaction dilute the reaction mixture 4 times in water before
- Add 20 µL of Working Reagent to each well. Mix gently by tapping the license plate.
- Incubate for 30 min at room temperature for colour development.
- Measure absorbance at 600 – 660 nm (620 nm) in a plate reader. For 384-well plate assays, the procedures are the same, except that the volume of the standard solution and sample should be 40 µL and that of the working reagent should be 10 µL.
- Incubation time. The chromogenic reaction is complete in 30 min at room temperature. Read the OD values at 30 min.
- Precipitation can occur at high phosphate concentrations (>100 µM), or in the presence of high concentrations of e.g proteins and metals. If precipitation occurs, perform a serial dilution of the sample in H2O, run the test and determine the dilution factor of wells without precipitation. Repeat assays using diluted samples.
- Enzyme reaction buffer. Because any exogenous free phosphate interferes with the assay, it is important to ensure that the protein preparation, the reaction buffer and the laboratory products used in the assay it must not contain free phosphate. This can be conveniently checked by adding a working reagent to the buffer and measuring colour training.
- Elimination of liquids. The test mixture contains 0.4 M sulfuric acid. It is recommended that the waste liquid be neutralized with a volume equal to 1 N NaOH before removal.