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Description
Human UBE2L3 ELISA KitProduct Specification Usage Required experimental equipment: 1. Microplate reader (450nm) 2. High precision pipettes and pipette tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37C incubator 4. Distilled or deionized water Sample preparation and requirements: Tissue homogenization: Rinse the tissue with pre chilled PBS (0. 01M, pH 7. 4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and
Product Specification
| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample preparation and requirements: Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and mince the tissue. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis. Cell Lysis Buffer: Gently wash adherent cells with pre-chilled PBS, then trypsinize and harvest cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Wash the harvested cells three times with pre-chilled PBS and resuspend in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. Pre-Assay Preparation: 1. Remove the kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the gradient working solution of the standard: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0.10 ng/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 10 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 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 uses 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 Ubiquitin Conjugating Enzyme E2L3 (UBE2L3) capture antibody. 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 Ubiquitin Conjugating Enzyme E2L3 (UBE2L3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Ubiquitin Conjugating Enzyme E2L3 | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ubiquitin-conjugating enzyme E2C-binding protein E2L3, also known as UBCH7, is encoded by the UBE2L3 gene. As an E2 enzyme, UBE2L3 participates in the ubiquitination of target proteins for degradation. UBE2L3's role in the ubiquitination of NF-κB precursors has led to its involvement in several major autoimmune diseases, including rheumatoid arthritis, celiac disease, Crohn's disease, and systemic lupus erythematosus. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenate, cell lysate |
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4.8 ★★★★★
Based on 20 reviews
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Product Reviews
★★★★★ 5
Great buy
Our dog is so happy about this. He loves chasing a ball. We also love the fact that it is collapsible and only 12 inches. We travel a lot so it’s perfect for that. I’m enclosing a movie. I did about it for Amazon.
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Reviewed in the United States on February 10, 2026
★★★★★ 1
Very disappointed
Unfortunately I ended up being very disappointed with this purchase, after about two weeks of use, it just stopped performing. Not sure what happened but it just stopped releasing the ball and I don't know why, it had no flexibility left to it. I'm not doing a very good job of trying to explain why it quit working but suffice it to say, it became useless after a couple of weeks. Broke my neighbors dog's heart, lol.
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Reviewed in the United States on November 20, 2025
★★★★★ 5
This is a big ball.
Larger than I thought but still a great toy for a heavy chew pup, easy to toss and won't go under funiture. She has the same ball in a 5 inch size so I know it's quality and will last. Can't wait for Summer, will make a great yard ball.
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Reviewed in the United States on March 22, 2026
★★★★★ 5
Second one, first one lasted a good while
We leave it outside in the weather with solid results for our backyard dog, and the dog loves this thing. It's our go to toy for her, ... perfect for kick to fetch, tug of war, hide in the shirt, bump the human leg when it is wet, etc. ;) The first one we used and used and finally it started to show too much weathering, mildew, and finally breaking after like 1.5 years... we have extremely hot and wet weather here. When new though, these balls take a pounding. Our 50 pound dog can play tug of war to the extent we can pick her up while she holds onto the ball with her teeth. The ball doesn't flinch and the dog loves it. Kicking it is fun because you can give it a wallop and it won't go over the fence since the design absorbs a fair bit of the kick energy, but it is still enough for an adult or teen to send it about 1/4 an acre away. What else... oh the dog has a funny habit of finding the one spot that isn't an empty pentagon... as in the plastic is filled in there... and she likes to bit on that part especially during tug of war. I should also mention our dog literally breaks apart and sometimes eats sticks and twigs in the yard, can't be healthy, so a nice diversion is this ball that she will chew on once in a while without destroying it.
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Reviewed in the United States on July 10, 2022
★★★★★ 5
Absolutely love this brand of ball for my dog
I end up replacing these every couple years, yep that's right, years. My border collie will grab this ball in his mouth and then kick another ball around. Don't know why he has to have something in his mouth to play soccer, but he is unique. He managed to tear one of these up when he was in his teething stage, but it took him months, and I took it away as soon as he managed to remove a piece of it. Otherwise, we usually lose these much more often than them getting destroyed through constant chewing, and I suspect heavy weathering outside for an extended period of time. Nowadays he doesn't chew on rubber nearly that often, he has developed his own picky preferences. This ball is his must have for playing around. He loves to drop it down the stairs and play fetch with himself. It's easy for him to grab, it isn't heavy so we don't worry about furnishing, walls, or family members when he is tossing it around. It is much more durable than most, with the exception of really heavy chewers/teething, even then it held up for a long time. As long as you are careful to remove it if they manage to chew through I would even try it out with teething puppies. The lawnmower is also this balls nemesis, it loses every time. If only I could convince the dog that he needs to stop sacrificing toys to the lawnmower :-)
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Reviewed in the United States on January 1, 2025