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Description
Human UCP1 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 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 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/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 a thermogenin (UCP1) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The color intensity is positively correlated with the thermogenin (UCP1) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Thermogenin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Mitochondrial uncoupling protein 1 (UCP1), also known as thermogenic protein and encoded by the UCP1 gene, is a mitochondrial carrier protein found in brown adipose tissue. It is used to generate heat through non-shivering thermogenesis and contributes quantitatively to offsetting heat loss in infants that would otherwise occur due to their high surface area-to-volume ratio. Its mediated heat production in brown fat uncouples the respiratory chain, allowing for rapid substrate oxidation at low ATP production rates. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.3 ★★★★★
Based on 13 reviews
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Product Reviews
★★★★★ 5
Very durable and long-lasting, great for strong chewers, gets rid of plaque in older dogs
I have a seven-year-old golden retriever who was never a strong chewer, she liked gnawing on toys and dental “bones” and destroyed shoes and a carpet when she was a puppy but it was never her favorite activity. Then I adopted two puppies of indeterminate breed, but likely Plott Hound and Lab, and they are aggressive chewers and want to chew on everything. I tried all kinds of teething things, as teething seemed to bother them more than any of the other four dogs I’ve had, and all were destroyed quickly, like they were treats instead of supposedly “long-lasting” chew bones. I tried these bones after I happened upon them on Amazon, and I could not be happier. My dogs contentedly gnaw on these for extended periods of time, obviously in heaven as they do so, and sometimes it’s the only way I have time to get anything done. When they were teething these definitely helped them with teething pain, and now they still want to chew on them every day. They can hold them easily and sometimes the puppies even share a bone due to the unique shape. My Golden had lots of plaque and nasty buildup on her teeth and was headed for needing dental cleanings under anesthesia, but now her teeth are sparkling white without a speck of plaque thanks to these. The bones have worn down over the course of eight weeks, but very gradually and not ever in pieces. Yes, the ends become prickly and are definitely not fun to step on, but I don’t let them chew on them outside to reduce them collecting dirt and rinse them every few days to get rid of whatever is on there. I have watched carefully for any signs of irritation in my dogs’ mouths or teeth issues, and have never seen any blood on the bones even after my dogs have determinedly chewed on them for an hour. They LOVE them, and because they are the only bones I have found that last longer than five minutes for my aggressive chewers, hold their attention for an extended period of time, and get rid of all the plaque on my older dog’s teeth, I HIGHLY recommend them.
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Reviewed in the United States on January 26, 2021
★★★★★ 4
Long lasting
Works great they last a long time my dog love them. We have three of them. Always getting them out to play with. Great product for the price.
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Reviewed in the United States on January 13, 2026
★★★★★ 5
Lasts forever/Great Investment!
Sadie is still chewing on the one I bought a month ago!? Definitely the best investment in a dog bone. Most I’ve ever found are gone in an hour ??
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Reviewed in the United States on May 18, 2026
★★★★★ 5
I love this and so does my dog
I got one of these, to try out on my super chewer pit mix, back in early April, it's now the end of June and she's still chewing it every day, almost all day! It has gotten smaller as she's chewed it but it's not broken or splintered or hurt her teeth! Just make sure to get the right size for the dog. Small dogs maybe the smaller ones but a big girl like mine needs the large or bigger. Idk if she gives a darn about the taste or smell of it but she loves chewing on it, and hasn't felt the need to chew my shoes or wtfe since she got this. I like it so much I got more for my friend who also has a few super chewers and she gives good reports on them too. Her chewer dogs have stopped destroying her house in favor of these chews. Just try not to step on them in the dark, cause that s#!t hurts, BAD! Lol especially if you step on the chewed parts, where its rougher. The attached Pic is showing how badly she's chewed it in almost 4 months! The chewing calms her anxiety and gives her something to focus on when she's too excited, especially late at night. I will be getting more of these for her. even thinking about trying other shapes to see if they hold up as well as the wishbone shape does. She's an adult dog so idk if these would be OK for puppies, as it might Crack their teeth.
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Reviewed in the United States on June 27, 2024
★★★★★ 5
My Dog's Favorite Bone! No staining or smells on carpet. AWESOME COMPANY! Will Support You--RISK FREE PURCHASE!
Benebone rocks! It is pricey for sure, but these are superior to nylabones in my opinion. The flavor is there that you can smell when you sniff the actual toy, but it does not stain or make the carpet smell.
It will slowly wear down, but it does not result in sharp edges. Does keep my puppy's attention and does withstand her chewing. She chews on this toy every day!
NOTE: I did have a problem with a previous order from Benebone. The bone broke almost immediately. The company immediately contacted me and sent me TWO replacements. The replacements lasted for a long time, and neither replacement broke like the first one; I think I just got a bad one with an air bubble or other defect. I would recommend watching your puppy/dog very carefully when you first give this toy. If it holds up well after about an hour or two of chewing, it will be just fine and you'll get many hours from it (my dog must have over 10 hours of just chewing on it). If it breaks, pull it away and contact the company! They were receptive and extremely supportive! When you have a dog you really love and is a part of the family, you really want a company that supports you both. Benebone is that company. I have recommended this bone to make co-workers and friends (I think I have had approximately 7 or 8 people buy this bone because of me), and they have all purchased the bone and it has been a HUGE hit success.
So it is risk free! Buy this bone for your pup or bone and see how it goes! If you don't like it or your pup doesn't use it, just contact the company and see what they will do for you.
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Reviewed in the United States on March 12, 2017
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