Description
Urokinase Assay Kit (BA0229) (BA0229)
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 Assay Kit (SKU: BA0229) provides a convenient fluorimetric method to measure urokinase activity in biological samples. In this assay, the fluorimetric substrate reacts with urokinase so that the increase in fluorescence at λex/em = 380/450 nm is directly proportional to enzyme activity. The homogeneous, non-radioactive assay is completed within a 15-minute reaction time and can be readily automated for high-throughput analysis. It provides a sensitive and accurate means of quantifying urokinase in serum and urine.
| Product Name: | Urokinase Assay Kit (BA0229) |
| SKU: | BA0229 |
| Detection Method: | Fluorimetric determination at λex/em = 380/450 nm |
| Detection Range: | 0.04 - 30 U/L urokinase in a 96-well plate assay |
| Sample Type: | Biological samples (e.g. urine and serum) |
| Species Reactivity: | All |
| Assay Time: | 15 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 assay for urokinase activity. The fluorimetric substrate reacts with urokinase so that the increase in fluorescence at λex/em = 380/450 nm is directly proportional to enzyme activity, with a linear detection range of 0.04 - 30 U/L urokinase in a 96-well plate assay.
- Safe. Non-radioactive assay.
- Fast. Assay is completed within a 15 minute reaction time.
- Sensitive and accurate. Linear detection range 0.04 - 30 U/L urokinase in a 96-well plate assay.
- Convenient and high-throughput. Homogeneous “mix-incubate-measure” type assay. Can be readily automated to assay thousands of samples per day.
- For quantitative determination of urokinase activity in 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 | Equilibrate all components to room temperature and briefly centrifuge tubes before opening. The Working Reagent (WR) should be prepared fresh for each assay run. |
| 2 | Prepare the enzyme in an enzyme buffer, e.g. 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.08% BSA, when required. |
| 3 | Sample preparation: serum can be assayed directly; urine should be diluted 2-fold or higher in water prior to the assay run. |
| 4 | Standard preparation: prepare a 50 µM Premix by combining 5 µL 2.5 mM Standard and 245 µL Assay Buffer, then dilute standards in a black flat-bottom 96-well plate to 50, 30, 15 and 0 µM as per the table. |
| 5 | Transfer 10 µL of each sample to separate wells of the plate. |
| 6 | Prepare enough WR for all sample wells by mixing 5 µL Substrate and 90 µL Assay Buffer per well. |
| 7 | Add 90 µL WR to all sample wells. Tap plate to mix and incubate for 15 min. Measure fluorescence intensity at λex/em = 380/450 nm. |
Subtract the blank value (Standard #4) from the standard values and plot ∆F against the standard concentrations to determine the slope (µM⁻¹). Urokinase Activity = [(F_Sample – F_Blank) / Slope] × [Reaction Vol / (t × Enzyme Vol)] × n (U/L), where t is the reaction time (15 min), Reaction Vol is 100 µL, Enzyme Vol is 10 µL and n is the sample dilution factor. Unit definition: 1 U of urokinase catalyses the conversion of 1 µmole of Substrate per min at room temperature and pH 7.4. If sample activity exceeds 30 U/L, dilute samples in enzyme buffer and repeat the assay.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| 2.5 mM Standard | 40 µL | -20°C |
| Substrate | 600 µL | -20°C |
