Description
Urokinase Inhibitor Screening Assay Kit (BA0228) (BA0228)
Urokinase plasminogen activator (urokinase, uPA) is a key serine protease involved in the degradation of the extracellular matrix that catalyses the conversion of plasminogen to active plasmin. It acts as a thrombolytic agent to break up blood clots and, when over-expressed, has been reported to influence the growth of certain malignant tumours such as breast and prostate cancers. The Urokinase Inhibitor Screening Assay Kit (SKU: BA0228) provides a convenient fluorimetric means to screen for potential urokinase inhibitors. In this assay, the fluorimetric substrate reacts with urokinase and an inhibitor will decrease the fluorescence at λex/em = 380/450 nm. The homogeneous, non-radioactive format is completed within a 30-minute reaction time and can be readily automated for high-throughput screening. Please note that the enzyme is not supplied; an enzyme of interest is required for this inhibitor assay.
| Product Name: | Urokinase Inhibitor Screening Assay Kit (BA0228) |
| SKU: | BA0228 |
| Detection Method: | Fluorimetric determination at λex/em = 380/450 nm |
| Detection Range: | Inhibitor screening assay for urokinase |
| Sample Type: | Compounds that affect urokinase activity |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped at room temperature. Store all components at -20°C upon receipt. |
| Shelf Life: | 12 months after receipt. |
| Shipping: | Room Temperature |
A quantitative fluorimetric inhibitor screening assay for urokinase. The fluorimetric substrate reacts with urokinase, and the presence of an inhibitor decreases the fluorescence measured at λex/em = 380/450 nm, allowing potential inhibitors to be evaluated and ranked.
- Safe. Non-radioactive assay.
- Fast. Assay is completed within a 30 minute reaction time.
- Convenient and high-throughput. Homogeneous “mix-incubate-measure” type assay. Can be readily automated to assay thousands of samples per day.
- For evaluation of drugs and screening potential inhibitors of urokinase.
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 | Equilibrate all components to room temperature and briefly centrifuge tubes before opening. The Working Reagent (WR) should be prepared fresh for each assay run. Note: enzyme is not included; an enzyme of interest is required. |
| 2 | Prepare the enzyme in an enzyme buffer, e.g. 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.08% BSA. Experimentally determine the optimal amount of enzyme per well if using an enzyme other than native human urokinase plasminogen activator. |
| 3 | Dissolve test compounds in a solvent of choice (e.g. DMSO); first test the tolerance of the solvent by the enzyme. Amiloride hydrochloride may be used to generate a reference IC50 curve. |
| 4 | Transfer 5 µL of Enzyme into separate wells of a black, flat-bottom 96-well plate. Reserve one well for the No Inhibitor Control and one for the No Enzyme Blank: add 5 µL Enzyme to the Control and 5 µL Assay Buffer to the Blank. |
| 5 | To the Control and Blank wells, add 25 µL of the solvent in Assay Buffer in which the test compounds are dissolved. |
| 6 | To the remaining Enzyme-containing wells, add 25 µL of the test compounds. Tap to mix and incubate for 10 min at room temperature to allow the inhibitor to block enzyme activity. |
| 7 | Prepare WR by mixing 5 µL Substrate and 95 µL Assay Buffer per well. Transfer 90 µL WR to all wells. Tap to mix and incubate for 30 min at room temperature. |
| 8 | Read the fluorescence intensity at λex/em = 380/450 nm. |
| 9 | 384-well format: incubate 5 µL Enzyme with 15 µL Test Compounds for 10 min, then add 60 µL WR. |
Enzyme Activity % = (F_Compound – F_Blank) / (F_Control – F_Blank) × 100%, where F_Compound, F_Control and F_Blank are the fluorescence values of the test compound, Control and Blank wells at 30 min.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| 10 mM Inhibitor | 40 µL | -20°C |
| Substrate | 600 µL | -20°C |
