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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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4.4 ★★★★★
Based on 23 reviews
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Product Reviews
★★★★★ 5
loved this book
Format: Kindle
On my first read. This book is a lot to digest. I love it though and it was worth the purchase.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 26, 2024
★★★★★ 5
This was a graduate thesis!
Format: Paperback
Imagine how useful academia could be if this kind of writing was accepted at the doctoral level these days! Here is a concise analysis of the problem with plausible solutions. Not what seriously more than 99% of all academic writing in sociology, literature, the art, comparative literature, and anthropology hold as a gold standard today; today you must obfuscate your pointless observations with academic jargon, citation and reference to others who have made no real observations, and fighting racism by supporting a new way of talking in order to maintain the existing inequitable systems.
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Reviewed in the United States on February 8, 2021
★★★★★ 5
A Riveting Read!
Format: Kindle
Come along for the page-turning tale of young Hannah, a brave young woman who just wants to do good in a time of madness. I read this in one sitting! Highly recommended for anyone interested in historical fiction. This would also make a wonderful accompaniment for school-age curriculum in grade 5 or above.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 14, 2025
★★★★★ 5
Great Book!
Format: Paperback
This book was a great read. So well written, and keeps you on your toes the entire time. I overall really enjoyed reading about Hannah's experience, and I could not put the book down because I was so eager to find out what happened to her! The author(s) clearly put a lot of time into researching the Salem Witch Trials and creating a book that has an accurate (and fact-based) representation of the historical event. I wish this book had been around when I was in elementary school or middle school, as I know my younger self would be in awe of Hannah's story and her unwavering bravery/compassion. Overall, a great read that I would highly recommend to others.
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Reviewed in the United States on January 5, 2021
★★★★★ 4
**Actual Rating 4.5** Highly enjoyable read!
Format: Kindle
Overall
This is a very well written fast paced read. Hannah is an excellent character with a strong moral compass. It has a bit of a slow start but picks up very quickly. It’s historical references are fantastic and the setting was beautifully drawn. It’s appropriate for the age range and even endeavors to show how actions have consequences. However, there were a lot of unresolved threads and even the outcome didn’t sufficiently demonstrate much of the short story strived to demonstrate.
That being said this short story read was well worth the time I very much enjoyed this little read. Best part is there are historical references. This is not the first iteration of the Salem Witch Trials, typically the story goes in one of two directions, where it was true (there were witches) or faked (girls on a power trip). This has been repeated through history and despite the excitement at the prospect of witches in 1692 Salem, the latter and the premise of this story is far more plausible.
Rating
4.5 (rounded down)
A strong educational and historical novella that’s setting and characterization leave a positive impression.
Where did I copy?
Amazon.
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Reviewed in the United States on February 12, 2021