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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 1613 reviews
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Product Reviews
★★★★★ 4
Immediate results
Item Package Quantity: 1
Very easy to apply. Impressive immediate results. However not yet sure if the shine will last. It’s only been 48 hours but so far it’s great and definitely worth the money. I give it 4 stars for now. If the shine lasts it deserves 5 stars.
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Reviewed in the United States on March 22, 2026
★★★★★ 3
Good…not great
Item Package Quantity: 1
Worked decently. Took care of the light fog but didn’t quite do the job on the center of the lights
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Reviewed in the United States on February 26, 2026
★★★★★ 5
It does work but
Item Package Quantity: 1, Item Package Quantity: 1
I recently bought a small general purpose utility vehicle. Because it was a tow along vehicle behind a motorhome since it was new it is in remarkable condition. That is except for the headlights. They actually aren’t yellowed with age but cloudy from exhaust fumes from the motorhome. Such a haze is hardened and doesn’t even scratch with a fingernail so I had very low expectations for this vehicle.
WHAT YOU GET
I looked at the listings for all of the famous brands, I chose this one primarily on price, after all I only bought this vehicle for quick errands around the neighborhood. I accept responsibility for not reading every word of their ad, but because I have used many well-known brands of headlight restorer in the past I know that most of them have enough so you can do a couple of vehicles at least. So I was completely not expecting getting three small packets of wipes. That’s it. It can be used only one time and now compared to the kits with bottles that can do several sets of headlights that cost twice as much I see this one is a not such a good value as I thought. And I never buy anything that comes as a wipe, usually by the time I get them they are already dried up, which makes them useless, that’s why they always have an expiration date. I changed my plans and went to work on the Jeep immediately to see if I could make those headlight lens covers usable (they look like sealed beams but the lenses are meant to protect the headlamps).
YES NO MAYBE
I can’t blame the ad, after reading it again they do state that these are only single use wipes, which means if you start a project you have to finish immediately. Hint: never use wipes on a hot day, they will start drying up as soon as you open the envelope and if the day is hot enough you might not even get through the job before they are totally dry. If I knew how these differed from the well-known brand names I would have made the choice to buy something in a bottle so I can use a real cleaning cloth and re-wet it if necessary. Did these clean my headlamp lenses? As a shopper all you want to know is yes or know, did it work and am I satisfied. The conclusion is they exceeded my expectations. The application process could not be easier, and note that well when some random person approaches you at a gas station and offers to clean your headlights, these are so easy my great-grandmother could apply them. You wipe the lenses with envelope #1, not rub, not scrub, just a simple wipe to make sure you get all of it. I was able to also do my clear lens fog lights which were equally crusted over. Make sure you dry them with a paper towel then let them air dry for a little while. I’m not sure what you would do if you live in an extremely high humidity area, and the manufacturer doesn’t address that. It was a dry day for me so I gave it fifteen minutes. Then you wipe the entire lens with packet #2 – again I was able to do two headlights and two fog lights with one swab. My headlight and fog lenses instantly turned crystal clear like they looked the day the car was made. I should take away a point because I didn’t expect this kit to be only three small envelopes with wet wipes in them but they don’t try to pretend otherwise and results are 100% of why I rated them a full five stars.
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Reviewed in the United States on October 16, 2024
★★★★★ 5
Good quality
Item Package Quantity: 1
My car is old this product does a very good job.
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Reviewed in the United States on May 3, 2026
★★★★★ 5
It really works!
Item Package Quantity: 1
This worked exactly as it says, and turned what looked like an old vehicle to a shiny new one. The only ugly thing about the car was the cloudy headlights. Everything else looked great. It really ages a car when it has cloudy headlights. There was no rubbing or buffing, just easy wiping and drying in one more wipe. The video was very helpful on the website.
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Reviewed in the United States on February 14, 2026