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Description
Mouse uPA ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect specimens using EDTA or heparin as anticoagulants and centrifuge them at 1000×g for 15 minutes at 2-8℃ within 30 minutes of collection. The supernatant can be tested or stored at -20℃ or -80℃, but repeated freezing and thawing should be avoided. Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant, and test. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the standard gradient working solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water (concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C. 2. Sample addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate. This will reduce the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculating experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against Plasminogen Activator and Urokinase (uPA). After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Plasminogen Activator and Urokinase (uPA) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Plasminogen Activator, Urokinase ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Urokinase-type plasminogen activator (uPA), also known as urokinase, is a serine protease. Produced in the kidneys, it directly activates plasminogen to convert plasmin. It is the first generation of natural thrombolytic drugs extracted from urine. Urokinase protein was discovered by McFarlane and Pilling in 1947, but remains unnamed. Initially isolated from urine, it is also present in blood and the extracellular matrix of many tissues. The enzyme's primary physiological substrate is plasminogen, the inactive form (zymogen) of the serine protease plasmin. It directly acts on the endogenous fibrinolytic system, catalyzing the cleavage of plasminogen into plasmin. Plasmin degrades not only fibrin clots but also circulating fibrinogen, coagulation factors V and VIII, thereby exerting its thrombolytic effect. It also increases vascular ADPase activity, inhibiting ADP-induced platelet aggregation and preventing thrombosis. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, and other biological fluids |
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4.2 ★★★★★
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★★★★★ 5
Blue ball, my dog likes blue.
Just a ball right? Evidently not! My dog won’t chase an orange chuck it ball or the glow in the dark ball- only the blue one 🤷🏼♀️
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Reviewed in the United States on May 9, 2025
★★★★★ 3
I wanted a blue ball
It's great. It just wasn't blue.
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Reviewed in the United States on January 5, 2026
★★★★★ 5
its a great ball
works well
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Reviewed in the United States on July 8, 2025
★★★★★ 5
Love this set!!!
Size: Medium (2.5"), Style: Fetch Pack 1
I was trying to figure out which of the many different balls this company offers, and I was getting frustrated but then i was SOOOO happy to see they had multiple sets that had multiple of the different balls they offer!! I bought this set and the other set they offer, but this one was my favorite and I think my dogs too! The orange ball works great and bounces just high enough that it doesn’t clear our patio wall, but enough for him to go and jump up to retrieve. But the glow in the dark ball is his favorite! Both me and my pomsky were blown away how we brought the glow in the dark ball outside just as the sun was going down, and when we brought it back inside it was BRIGHT AND GLOWY!!! Even with the sun going down and not much light left!! He loves to play with it in the house after it’s all glowy from the sun. Sometimes we even glow it up under a light inside the house if it’s dark outside, and he will play with it outside in the dark haha he loves it!!! And I think it’s pretty cool too!! All of the balls this company overs are VERY durable! We realized early on that we cannot give our dog regular tennis balls cuz his favorite thing to do is tear the outside fabric off!! So we were trying to figure out what company has the best balls for dogs so they can’t sit there and try to pry them apart, and we found this company!! We have already bought from them about 3 times and all of their balls are durable and bounce how they say they will bounce!! They have the super bouncers and the glow in the dark one and the one with angles that will bounce in different directions and the ones you can throw REALLYY far! Overall, all the balls from this company are very durable, bounce really good, and keep my dog occupied without allowing him to sit there and tear it apart!!! Which i love cuz I do not like when he stops playing with us to sit there and try to take off the fabric, cuz then he could swallow it! So always buy the balls from this company!!! They are the best and most durable!!!! Also plenty soft for your dogs mouth so they can chew on it a bit and it won’t hurt their teeth/gums!
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Reviewed in the United States on July 28, 2024
★★★★★ 5
Good sturdy balls
Size: Medium (2.5"), Style: Fetch Pack 2
My dogs love balls! Unfortunately, they tend to chew them up very quickly. My Belgium Malinois chews on this all the time, and it has held up pretty well. Good option for chewers
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Reviewed in the United States on May 17, 2026