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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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4.1 ★★★★★
Based on 1316 reviews
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Product Reviews
★★★★★ 4
Looooves her crazy new ball
Color: Orange, Color: Orange
Our 90lb mastiff/boxer mix Indie is very playful, curious and energetic. She needs toys that engage her and keep her busy. This ball does the job!
We’ve had it for less than a week. Here’s what I love and what could be better:
PROS:
• She dog loves it. She whines for it. She gets absurdly excited when I get it out. She’s a bit of an anxious dog, but had zero problems with it inexplicably coming to life.
• It lasts a while on a single charge, so it keeps her busy for a good long time. The longest we’ve used it at a stretch is an hour.
• It’s pretty durable. She’s a super chewer (destroyed every “non-destructible” toy we’ve purchased for her), but the actual ball is hard plastic. She gnaws on it and it gets scratches, for sure. But she’s not going to chew it to pieces.
• On carpet, it’s reasonably quiet.
• It comes with a replaceable silicone button.
• It’s easy to charge…just pull off the silicone button and the charging port is inside.
• The silicone button is flush with the ball’s hard plastic surface, so there’s no way for her to pull the silicone button out and swallow it or get to the charging port.
CONS:
• Cover is worthless if your dog is a chewer. It goes on like an envelope with a Velcro closure. We took it off immediately.
• Without the cover, it’s very noisy on hardwood floors. To us, it’s worth it. (Though, I work from home, so I might feel differently after the holiday vacation.)
• She’s an XL dog, so she can fit the whole ball in her mouth easily and accidentally chomp down on the button and turn the ball off. Not a big deal, just something to know if you’ve got a Lab or larger dog.
• With extended playtime, the two halves can come unscrewed, especially with a large dog who uses their paws/jaws on it a lot. Thankfully, it makes a loud buzzing noise when this happens, so we know we need to take it from her and screw it back together. Is this a potential danger? With the rechargeable battery inside, I’d say yes. But we also don’t let her play with this toy without supervision. So keep that in mind.
Bottom line:
It has its drawbacks, but it keeps her entertained and seems like it should last her at least 6 months with supervised play. Which is honestly REALLY good when we’re talking about Indie, Destroyer of Toys. I’d buy it again.
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Reviewed in the United States on December 31, 2024
★★★★★ 3
Difficult/impossible to unscrew plastic cups (in order to recharge the vibrating/blinking device)
Color: Orange
Generally, I don't leave feedback on items purchased. However, in this case, I feel compelled to do so in hopes the mechanical engineers (of this product) will take my input to heart and ultimately modify it.
First the pros...
- I have 2 high-energy dogs (Jack Russell Terriers) who love their toys (e.g., chewing bones, tug-of-war, etc.) but usually get bored with them rather quickly.
- This interactive PetDroid ball, however, keeps them busy for a long time.
- In fact, my smaller dog (Max) knows its storage location and sits (as early as 6 AM) in front of the cabinet so that I give him "his ball". It keeps him busy for up to 1-2 hours at a time.
- Max is literally addicted to the PetDroid ball... his determinant play with it makes me laugh all the time.
Now, the cons...
- After several days of play, the exterior looks chewed up quite a bit. That's no problem though.
- Unfortunately, the interior "threads/grooves" of the two plastic cups (for closing/fasting) are very tiny.
- Thus, after an hour-long hard play, it is almost impossible to unscrew the 2 plastic cups in order to recharge the interior device.
- It appears the 2 cups somehow come off track and get realigned from the inside.
- At that point, I cannot unscrew the PetDroid ball in order to recharge the device.... and without the blinking/vibration, this toy is no longer fun for play.
- Ultimately, I had to place the bottom half of the ball into a vise and then use a plumbing wrench to untwist it.
Proposed Solution:
- Increase the size of the plastic cups' interior threads/grooves so that they won't get realigned/come off track.
- This would mitigate the current dilemma of NOT being able to unscrew the ball in order to recharge it.
In my view, this should be a relatively simple fix and significantly improve the durability of this product.
Thank you for your time.
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Reviewed in the United States on February 4, 2022
★★★★★ 5
Good fun and entertaining!
Color: Orange
A really fun interactive toy for your dog! It's pretty erratic and really gets him chasing it. The included soft cover was kinda hard to install and then it's hard to turn it off and on with the cover on, I feel like the cover didn't really improve it at all, sure it's louder without the cover but I feel like dog also likes it that way, he likes loud squeaky toys and this thing making a bunch of noise is appealing to him. We have mostly hardwood floors so it really gets moving, I'm sure on carpet it would be a bit quieter and maybe even a bit slower to move around but on hard smooth surfaces this things can really get zipping around abd get some speed to make him chase it. Overall a fun and entertaining toy.
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Reviewed in the United States on January 27, 2026
★★★★★ 5
Awesome
Color: Orange
Awesome Toy . My Jack is crazy over it. She usually get bored with others, but this toy still has her attention after a couple of weeks Runs a long time on a short charge. 👍🏻
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Reviewed in the United States on May 30, 2026
★★★★★ 2
Not the Fetch-Fest I Hoped For
Color: Orange
I had high hopes for the PetDroid Interactive Dog Ball, especially with the newly upgraded features and promises of durable, motion-activated fun. Unfortunately, it didn’t quite live up to expectations for my furry friend. Here’s why I’m giving it 2 stars.
Promising Features, Lackluster Results
The idea behind this toy is fantastic: a durable, motion-activated ball that would entertain my dog without much effort on my part. The ball is supposed to roll around on its own, keeping my dog engaged and active. However, in practice, it just didn’t capture my dog’s interest.
Durability
I will give credit where it’s due: the ball is well-made and seems durable. It can withstand some rough handling, which is great for more aggressive chewers. Unfortunately, durability doesn’t mean much if your dog won’t play with it in the first place.
Motion Activation
The motion activation feature is hit or miss. While the ball does move around as advertised, it didn’t seem to move in a way that intrigued my dog. He gave it a sniff, watched it for a minute, and then walked away, unimpressed. I tried multiple times to get him interested, but he simply wasn’t having it.
Lack of Engagement
The biggest issue is that the ball just didn’t engage my dog at all. It might be more suitable for a different personality type or breed, but for my pooch, it was a total flop. He’s usually quite playful and curious, so I was surprised by his lack of interest.
Final Thoughts
Overall, the PetDroid Interactive Dog Ball might work for some dogs, but it was a miss for mine. If your dog is easily entertained by automated toys, it might be worth a try. However, based on my experience, I can’t wholeheartedly recommend it.
Pros:
Durable construction
Motion-activated as advertised
Cons:
Did not engage my dog
Motion activation wasn’t intriguing enough
Might be more suitable for specific dog types or personalities
If you decide to give it a shot, just be prepared for the possibility that your dog might not find it as entertaining as you hope. For us, it’s back to the drawing board for a more engaging toy.
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Reviewed in the United States on June 14, 2024
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