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Description
Human USP6NL 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 ultrasonically disrupted or repeatedly frozen and thawed. Finally, the homogenate is centrifuged at 5000×g for 5-10 minutes and the supernatant is collected 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 capture antibody against USP6 N-Terminal Like Protein (USP6NL). 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 intensity of the color is positively correlated with the amount of USP6 N-Terminal Like Protein (USP6NL) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human USP6 N-Terminal Like Protein ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | USP6 amino-terminal-like protein (USP6NL), also known as UUSP6 N-terminal-like protein, is a protein encoded by the USP6NL gene. It acts as a GTPase activator for RAB5A and RAB43. It is involved in receptor trafficking. In complex with EPS8, it inhibits EGFR internalization. It participates in retrograde transport from the endocytic pathway to the Golgi apparatus. It participates in the transport of Shiga toxin from early and recycling endosomes to the trans-Golgi network and is required for the structural integrity of the Golgi complex. | |||||||||||||||||||||||||||||||||
| 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 and other biological fluids |
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4.9 ★★★★★
Based on 16 reviews
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Product Reviews
★★★★★ 4
Not real sweet, but no sugar
I have been taking Metamucil with real sugar regularly for 20+ years. I became concerned about the amount of sugar I was consuming daily and cannot tolerate aspartame. So, I jumped to buy this when I saw Stevia. I quickly noticed that this version is not sweet. It’s sort of bitter. Also, the texture is different due to not having sugar granules mixed in. It also converts to a gel like texture quicker. I later noticed you use less of this product per serving than the one with sugar (read the label!). I have found the best way to take this is to very quickly (seconds) consume the serving after adding water. Don’t wait too long. I down it like taking a shot out of a shot glass followed quickly by a glass of water. After a while, I got used to the flavor. Overall, I am pleased I am consuming much less sugar and really don’t notice the change anymore.
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Reviewed in the United States on June 2, 2022
★★★★★ 5
This product is very important to your well being.
The Metamucil is everything I hoped for. If you’re concerned about your sugar intake then I suggest the one with Stevia. Yes the one with natural sugar tastes the best but once you graduate to 3 times a day that’s a lot of sugar. I would follow the directions. Start with once a day then after 3 or 4 days you’ll find twice a day works nice. I’m on my 4th week so a few days ago I started 3 times. I’m very pleased with the way I feel. Think about it, we take care of our cars better than ourselves sometimes. It’s so important to take care of your digestive system. Have a good day. 😁
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Reviewed in the United States on March 23, 2021
★★★★★ 5
Great flavor, less calories than real sugar, thickens very quickly.
I usually get the real sugar version of this rather than this one with Stevia. I have it every morning just to supplement my diet with fiber and the version with stevia is only 30 calories vs 80 for the one with real sugar. I mix it with unflavored protein powder in a shaker cup and this one thickens very quickly. So quickly that I add water after every quarter of the container that I drink and re-shake. Otherwise it gets so think it won’t even come out by the time I’m halfway through. Once I figured that out it’s no big deal but makes it so that I won’t take it in the car to drink on the go.
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Reviewed in the United States on October 26, 2019
★★★★★ 5
IBS C must have!
I have been suffering with OBS C for a very long time now and my doctor through Amazon Care (if you haven't downloaded that app yet, do it now) recommend that I take magnesium and the Align probiotic with breakfast and Metamucil in the evening before bed. I am finally feeling better and have not had any issues using the restroom! I recommend for sure. The taste is meh but I would rather down something that doesn't taste great as long as I feel better anyway!
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Reviewed in the United States on June 2, 2022
★★★★★ 5
Very functional for our family - We ordered a second one
Color: Black
We were happy to stumble on this paper towel holder after trying a few that just either didn’t look great and didn’t work well. We really like the black matte finish and the fact that the base is pretty heavy so you can actually tear off a paper towel without the whole thing toppling over. The spray/cleaner is an added bonus to have right there also. It’s awesome for craft time and cleanup after kids.
We purchased one for both our kitchen table and our rental property and they both function great and have been holding up very well. Happy with the purchase!
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Reviewed in the United States on March 15, 2024
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