Description
Malachite Green Assay (BA0164) (BA0164)
The Malachite Green Assay (SKU: BA0164) provides a rapid colorimetric method for determining free phosphate, based on quantification of the green complex formed between malachite green, molybdate and free orthophosphate. The rapid colour formation can be conveniently measured on a spectrophotometer or a plate reader between 600 and 660 nm. The non-radioactive colorimetric format has been optimised to offer superior sensitivity and a prolonged shelf life, with an innovative formulation that prevents reagent precipitation and removes the need for filtration before use. The assay is simple and fast, involving a single addition step, and can be performed in tubes, cuvettes or multi-well plates. It is well suited to high-throughput screening of enzyme inhibitors, with Z factors of 0.7 to 0.9 observed in 96-well and 384-well plates.
| Product Name: | Malachite Green Assay (BA0164) |
| SKU: | BA0164 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.02 - 40 uM phosphate |
| Sample Type: | Enzyme reactions and biological samples containing free phosphate |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 2500 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store the reagents and standard at 4 C. |
| Shelf Life: | 1 year |
| Shipping: | Room Temperature |
A rapid colorimetric assay for the determination of free phosphate at 620 nm. Malachite green, molybdate and free orthophosphate form a green complex measured between 600 and 660 nm. The single-addition mix-and-measure format is stable, requires no filtration and is robust and amenable to high-throughput screening.
- Very stable reagent that does not precipitate, requiring no filtration before assays
- High sensitivity and a wide detection range: as little as 1 pmole of phosphate and a useful range of 0.02 - 40 uM
- Fast and convenient homogeneous mix-and-measure format
- Robust and amenable to high-throughput screening (Z factors of 0.7 to 0.9)
- Phosphatase assays (liberation of phosphate from peptide, protein or small-molecule substrates)
- Lipase and nucleoside triphosphatase assays
- Quantitation of phosphate in phospholipids, proteins and DNA
- High-throughput screening for phosphatase inhibitors
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Prepare enough Working Reagent by mixing 100 volumes of Reagent A with 1 volume of Reagent B (e.g. 5 mL Reagent A and 50 uL Reagent B). The Working Reagent is stable for at least one day at room temperature; bring it to room temperature before use. |
| 2 | Verify that enzyme preparations and assay buffers are free of phosphate by adding 20 uL Working Reagent to 80 uL sample; blank OD values at 620 nm should be below 0.2. |
| 3 | Prepare a 40 uM phosphate premix (40 uL of 1 mM standard in 960 uL water or reaction buffer) and dilute to 40, 32, 24, 16, 12, 8, 4 and 0 uM. Pipette 80 uL of each standard in duplicate into wells of a clear-bottom 96-well plate. |
| 4 | Transfer 80 uL of each test sample into separate wells. For ATPase or GTPase assays, keep the ATP or GTP concentration below 0.25 mM, diluting the reaction mixture if necessary. |
| 5 | Add 20 uL Working Reagent to each well and mix gently by tapping the plate. |
| 6 | Incubate for 30 min at room temperature and measure absorbance at 600-660 nm (620 nm). A 384-well format is also supported using 40 uL sample and 10 uL Working Reagent. |
Plot the optical density at 620 nm against phosphate standard concentration and determine sample phosphate concentrations from the standard curve.
| Component | Quantity | Storage |
| Reagent A | 50 mL | 4 C |
| Reagent B | 1 mL | 4 C |
| Standard (1 mM phosphate) | 1 mL | 4 C |
