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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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4.2 ★★★★★
Based on 2341 reviews
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Product Reviews
★★★★★ 5
Amazing read!
Format: Kindle
I loved this book so much! I might be a little biased because I loved the first one so much and I’ve been dying for Ana to get happy ending ever since, but it was definitely a good book all around.
Ana and Rachel were absolutely perfect for each other. They have the kind of opposing personalities that fit together like a puzzle. Where Rachel was a little more reserved and polished, Ana was more outgoing and messy. They didn’t clash, they completed each other. I’m not going to lie, love that they’re a doctor and a lawyer because they’re giving sapphic power couple.
I also loved all of the supportive family members and best friends in this book. Stella and Charlotte were super cute and I hope we get to see them again in a later book.
I thoroughly enjoyed this book and I’d recommend it to anyone looking for an opposites attract, doctor/lawyer romance. This is the second book that I’ve read from Morgan and it seems like she doesn’t miss.
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Reviewed in the United States on June 18, 2025
★★★★★ 4
Sapphic Lawyer and Doctor
Format: Paperback
I loved this book… which is why I was very disappointed when the conflict was “solved” so easily and lowkey brushed past. We are waiting for this secret to come out that Rachel has been holding onto. There’s obviously conflict of interest with Ana and Rachel. But the way they solved it? I didn’t feel like it was realistic and it felt like both women were like, “Okay, that’s over, everything is kosher now(pun intended?). It honestly prevented me from giving it 5 stars. The ending seems so rushed.
But other than that, I loved both characters! I loved learning about Jewish culture and loved how the author portrayed it. Both MCs are fleshed out, in depth characters. The author had a good balance of humor
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Reviewed in the United States on September 11, 2025
★★★★★ 5
Enjoyable Doctor, Lawyer Romance
Format: Paperback
A very enjoyable and engaging story. The medical and legal aspects of this story was very fascinating. I never get tired of reading romances that are about those troupes. The romance between the main characters was very sweet with not A lot of drama or angst. Sometimes I prefer A story with less drama and angst. I like when characters in A story can be reasonable and work out their problems without all the drama. Ana and Rachel are those characters. There's A few witty moments in this book that made me smile and laugh out loud A few times throughout this story. The supporting characters were written well and helped to make this an even better reading experience. I will definitely be on the lookout for more books by this author.
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Reviewed in the United States on June 11, 2025
★★★★★ 4
So good
Format: Paperback
When your one-night stand turns out to be the surgeon that you subpoenaed for a malpractice lawsuit.
Ana, an unappreciated lawyer, has been passed up for promotion to her male colleagues yet again. A malpractice lawsuit win could be what she needs to finally get the promotion she’s worked so hard for. Rachel, a cardiac surgeon who goes above and beyond for her patients, is subpoenaed to testify in a malpractice lawsuit. Arriving at the law firm for her deposition, Rachel discovers that the lawyer for the family who filed the lawsuit is Ana Mendez. As in Ana, her one-night stand.
I was excited to get my hands on this one! I loved that Ana and Rachel were so different from each other in personalities, looks, backgrounds, and religions. The differences made them unique and interesting to me. The banter was well-written, and I loved how feisty they were towards each other in the beginning. Was the dirty talk unexpected? Very much so. I was not prepared for that or for how hot things had gotten. And while I did enjoy the spice, I would’ve liked for there to have been more communication about the conflict too. The references to The Golden Girls and The Sims? Oh, I totally geeked out over that! Overall, it was a good sapphic romance that I’m sure others will enjoy.
I’d like to thank NetGalley and Bold Strokes Books for the arc. This is my voluntary review.
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Reviewed in the United States on June 12, 2025
★★★★★ 4
A Conflict of Interest Worth Taking
Format: Paperback
This is the second book by Morgan Adams, and I thoroughly enjoyed it! One of the main characters, Ana Mendez, appears in Adams's first book, but that is not a requirement for reading this story about her and Dr. Rachel Cohen. One of the tropes in this book is one night to forever, and I enjoyed the time it took for the two characters to re-meet and figure out their issues.
I thought their relationship developed well on the page. It was believable, had some humor, and was QUITE spicy. I liked each character's relationships with friends and family, too; they helped to make Ana and Rachel more than just two women who are well-established in their careers.
A Conflict of Interest is a fascinating medical/legal story alongside a romantic one. I would recommend it to readers who want to read about two competent and mature adults who have to navigate their careers as well as a romance.
Thank you to the author and the publisher for the opportunity to read and review the arc of this book!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 11, 2025