Description

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. 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 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

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 1.6 pmoles of phosphate and useful range between 0.02 mM and 40 mM phosphate.

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.

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

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: 2,500 assays in 96-well plate

Reagent A: 50 mL    Reagent B: 1 mL    Standard: 1mL 1 mM phosphate    

Storage conditions. 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 refer to Material Safety Data Sheet for detailed information.

Procedure using 96-well plate

Reagent Preparation. Each assay requires 20 mL Working Reagent. Prepare enough Working Reagent by mixing 100 vol of Reagent A and 1 vol of Reagent B (e.g. 5 mL Reagent A and 50 mL Reagent B). Working Reagent is stable for at least 1 day at room temperature.

Important: The reagent 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 done by adding 20 μL of the Working Reagent to 80 μL 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 40 mM phosphate by pipetting 40 mL 1 mM phosphate standard to 960 mL distilled water or enzyme reaction buffer. Number the tubes.