Description
Kinase Assay Kit (Fluorometric) (BA0125) (BA0125)
This Kinase Assay Kit (Fluorometric) (SKU: BA0125) provides a simple and rapid method for assaying kinase activity and for high-throughput screening of kinase inhibitors. Kinases, also known as phosphotransferases, transfer phosphate groups from ATP to specific substrates and regulate the majority of cellular processes, making them important drug targets. In this homogeneous microplate-based assay the kinase is incubated with a single working reagent in which the ADP produced is enzymatically converted to ATP and pyruvate, which is quantified fluorimetrically at λex/em = 530/590 nm. The assay is non-radioactive, sensitive and robust, tolerating up to 300 µM ATP and 10% dimethyl sulfoxide. It is readily automated for tens of thousands of assays per day.
| Product Name: | Kinase Assay Kit (Fluorometric) (BA0125) |
| SKU: | BA0125 |
| Detection Method: | Fluorimetric (λex/em = 530/590 nm), ADP detection |
| Detection Range: | As low as 0.01 U/L kinase; tolerates up to 300 µM ATP and 10% DMSO |
| Sample Type: | Purified kinases, cells and other biological samples (user-supplied enzyme, ATP and substrate) |
| Species Reactivity: | All |
| Assay Time: | 10 minutes detection (plus user-defined kinase reaction, e.g. 30 minutes) |
| Kit Size: | 400 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all reagents at -20°C. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
The kinase reaction produces ADP, which is enzymatically converted to ATP and pyruvate. The pyruvate is quantified by a fluorimetric method at λex/em = 530/590 nm, and the signal is proportional to kinase activity. One reaction volume is 20 µL and the detection is a single working reagent.
- Safe, non-radioactive assay.
- Sensitive and accurate, quantifying kinase activity as low as 0.01 U/L.
- Homogeneous and convenient 'mix-incubate-measure' assay using a single working reagent and 10-minute incubation at room temperature.
- Robust and amenable to high-throughput screening, tolerating up to 300 µM ATP and 10% DMSO with Z' factors typically above 0.6, readily automated for tens of thousands of assays per day.
- Kinase activity assays and high-throughput screening for kinase inhibitors.
- ADP determination in cells and other biological samples.
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 | This kit is sufficient for 400 assays in 384-well plates (200 assays in 96-well plates); use black flat-bottom plates, bring all reagents to room temperature and run assays in duplicate. |
| 2 | Interference: thiols (β-mercaptoethanol, dithioerythritol, etc.) above 10 µM interfere with this assay and should be avoided. |
| 3 | Kinase reaction: users supply their own enzyme, ultra-pure ATP and substrate; set up a 20 µL reaction mixture containing the kinase, ATP and substrate in the provided Assay Buffer (pH 7.0) or a suitable kinase buffer, include a blank control with ATP and substrate but no enzyme, and incubate at the desired temperature for the desired time (e.g. 30 minutes). |
| 4 | Standards: prepare 900 µL of 10 µM ADP Premix by mixing 3 µL of the 3 mM Standard with 897 µL distilled water, dilute as described in the standard table and transfer 20 µL of each standard to separate wells. |
| 5 | ADP detection: prepare Working Reagent by mixing, per well, 25 µL Reagent A and 25 µL Reagent B, add 40 µL Working Reagent to each assay well, tap to mix and incubate at room temperature for 10 minutes. |
| 6 | Read fluorescence intensity at λex = 530 nm and λem = 590 nm. |
| 7 | For inhibitor screening, pre-incubate test compounds with the kinase for 10-30 minutes (using a known inhibitor such as staurosporine as a positive control and the compound solvent as a negative control) before adding ATP and substrate to initiate the reaction, then detect the ADP produced as above; the fluorescence decreases in the presence of an inhibitor. |
Kinase Activity (U/L) = (ΔF_SAMPLE / (Slope x t)) x (20 µL / Vol), where ΔF_SAMPLE is the fluorescence of the sample well minus that of the blank well, Slope is the slope of the ADP standard curve, t is the kinase reaction time (e.g. 30 min) and Vol is the volume (µL) of kinase added to the 20 µL reaction. If ΔF_SAMPLE exceeds (F_10µM ADP - F_H2O), dilute the enzyme in Assay Buffer, repeat and multiply the result by the dilution factor to ensure the rate is measured at less than 10% ATP conversion.
| Component | Quantity | Storage |
| Reagent A | 10 mL | -20°C |
| Reagent B | 10 mL | -20°C |
| Assay Buffer | 25 mL | -20°C |
| Standard (3 mM ADP) | 100 µL | -20°C |
