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Description
Human UbcH5c 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: Adherent cells should be gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells should be washed three times with pre-chilled PBS and resuspended 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. 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 UbcH5c capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and then to yellow by acid. The intensity of the color is positively correlated with the amount of UbcH5c in the sample. Absorbance (OD) is measured at 450 nm using a microplate reader to calculate sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human UbcH5c ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ubiquitin-conjugating enzymes, also known as E2 enzymes and less commonly as ubiquitin-carrying enzymes, perform the second step of the ubiquitination reaction, directing proteins for degradation by the proteasome. The ubiquitination process covalently links ubiquitin (a short protein of 76 amino acids) to a lysine residue on the target protein. Once a protein is tagged with a ubiquitin molecule, additional rounds of ubiquitination form a polyubiquitin chain that is recognized by the proteasome's 19S regulatory particle, triggering ATP-dependent unfolding of the target protein and its entry into the proteasome's 20S core particle, where proteases degrade the target protein into short peptide fragments for cellular recycling. | |||||||||||||||||||||||||||||||||
| 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 | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.7 ★★★★★
Based on 134 reviews
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Product Reviews
★★★★★ 4
A Nice Story
Format: Kindle
I love Omegaverse and its many stories. The book is a wonderful story. The characters are intriguing and are each unique. There were a few issues with repetitive phrases through out the book and a few misspellings but the biggest issue was the very painful slow burn. I feel if it weren’t so slow
It could have incorporated more content about each character. Overall a solid story though.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 25, 2025
★★★★★ 3
decent…but
Format: Kindle
This is my first book from this author, and while I do really love omegaverse stories, this one left a lot to be desired. This book is a hard-core slow, slow, slow burn: (75% or later in the book)
I will say that I finished it. Unfortunately, I struggled through a lot of it due to punctuation errors, and general editing that must have been looked over during the revision process.
I also feel like a lot of information was left out and there wasn’t closure regarding her parents. The dynamic of the pack before Lydia joined is a little confusing, the only person who showed affection towards Elias was the Head alpha. The other two seemed indifferent to him in an intimate relationship way. That doesn’t really scream cohesive pack to me.
I am hopeful that the next book in the series is better edited, and that the author decides not to use the same phrases over and over again. I’m pretty sure “honey-warm” was mentioned over 15 times when describing Elias’s scent. That got really old, really fast.
Overall, it was a decent book. Not sure I would read it again or recommend it until of these errors are remedied.
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Reviewed in the United States on April 26, 2025
★★★★★ 5
Definitely a comfort read
Format: Kindle
Just a comfy cozy nice long read. While this is definitely a SLOOOOOOOOWWWWW burn, this is how you do RH right; a nice long novel where you actually get to know the characters so you can feel more connected to them & the story, not to mention getting enough time to actually remember all their names haha. & they are all consent kings. Also I do so love me some double omega OV, siiiigh (heart eyes). I wish there had been juuust a bit more spice, like maybe just between Elias & others before MC was ready (made Elias feel a bit underdone or gimmicky without it), I did love this read. Low stress, comfy read. It could've used another round of editing for some minor errors & type issues but it wasn't bad enough to pull ne out of the story. Though I did giggle at 'knecks', thinking kuh-necks in my head. Overall I'd say 9/10
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Reviewed in the United States on March 28, 2025
★★★★★ 4
beautiful story
Format: Kindle
I absolutely love this book, however there is quite a bit repetition! The storyline makes up for it, a just skipped them. I like that this was a cozy read, it’s an omegaverse romance, but really barely read as OV, you get a little hierarchy and scent blockers, but if not for that you wouldn’t know. I love the romance aspect however, they take there time and move at her pace! A good read, with some errors, hopefully there is more to come!!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 13, 2025
★★★★★ 2
Nicole
Format: Kindle
I loved the concept of the story but it went no where painfully slow! There was so much repetition that I skimmed and only paid attention whenever I saw quotation marks. I need...yes we know what you need....said 125 times grew frustrating and tiresome! And what does progressive mean in this boom? There are 3 alphas and 2 omegas...is that different or is it bc of the 3 alphas and Lucian was the really alpha one. The sex was very disappointing too....very G rated in my spice level. There is no epilogue and I think it was like 80 something chapters! I should have read the reviews but the sample got me good! What does soren do for a living? Are the men sexual w eachother bc they did not show that in this story if you are worried about that. It was jus so
painstakingly slooowwwwww!!! I get why but dang! Just beware g rated and slow paced w alot of repetition!
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Reviewed in the United States on June 2, 2025
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