Description
Phosphate Assay Kit (BA0165) (BA0165)
The Phosphate Assay Kit (SKU: BA0165) provides a rapid colorimetric method for determining free phosphate, based on a proprietary malachite green formulation. The reagent forms a blue complex with free orthophosphate that 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 plates.
| Product Name: | Phosphate Assay Kit (BA0165) |
| SKU: | BA0165 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.4 - 50 uM phosphate |
| Sample Type: | Enzyme reactions and biological samples containing free phosphate |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 500 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store the reagent and standard at 4 C. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
A rapid colorimetric assay for the determination of free phosphate at 620 nm using a proprietary malachite green formulation. The reagent forms a blue complex with free orthophosphate 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 20 pmoles of phosphate and a useful range of 0.4 - 50 uM
- Fast and convenient single-reagent 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 | Bring the reagent to room temperature and shake it well before use. Ensure that enzyme preparations, assay buffers and lab ware are free of phosphate. |
| 2 | Prepare a 40 uM phosphate premix (40 uL of 1 mM standard in 960 uL water) and dilute to 40, 32, 24, 16, 12, 8, 4 and 0 uM. Transfer 50 uL of each standard in duplicate into wells of a clear-bottom 96-well plate. |
| 3 | Transfer 50 uL of each test sample in duplicate into separate wells. Enzyme reactions may be stopped directly by adding the reagent. |
| 4 | Add 100 uL reagent to each well and mix by tapping the plate. |
| 5 | Incubate for 30 min at room temperature for colour development. |
| 6 | Measure absorbance at 620 nm (600-660 nm). For cuvette assays, add 800 uL reagent to 400 uL sample and standards. |
Plot pmoles of phosphate against the optical density at 620 nm for the standard curve and use linear regression to determine the amount of free phosphate in the test samples.
| Component | Quantity | Storage |
| Reagent | 50 mL | 4 C |
| Standard (1 mM phosphate) | 1 mL | 4 C |
