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Description
Human RCN3 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 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 Reticulocalbin 3 (RCN3) 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 intensity of the color is positively correlated with the amount of Reticulocalbin 3 (RCN3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Reticulocalbin 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Reticulocalbin 3 (RCN3) is a protein-coding gene. Diseases associated with RCN3 include myopathy, distal myopathy 3, and neonatal respiratory failure. Gene Ontology (GO) annotations associated with this gene include calcium binding. An important homolog of this gene is RCN1. | |||||||||||||||||||||||||||||||||
| 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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4.4 ★★★★★
Based on 1444 reviews
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Product Reviews
★★★★★ 5
Make sure you get this brand. The look alikes are trash!
Very durable and fun! My Pitbull chews on this constantly and loves to have treats or peanut butter, put in the bottom of it as an extra treat. A friend of mine bought a similar looking toy that was destroyed by her pitbull in moments. This one is better quality and has stood the test of this power chewer. The rubber is very thick and he chews it constantly but it stays in tact. It is soft enough to squeak if he gets a hold of it just right, but that’s the rare treat. The size is perfect for my 80 pound boy. I am also able to throw these toys in his pool for him to dive for them. Definitely in great value for the price.
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Reviewed in the United States on March 19, 2026
★★★★★ 4
Indestructible, but maybe too much?
Color: Orange, Size: Medium
Not too enticing for my dog, but, it is definitely indestructible! He chews on it every so often, but less than I had expected. I think the material is so rock hard that it offers little incentive for him to chew on it.
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Reviewed in the United States on May 27, 2026
★★★★★ 5
Best I've Tried
Color: Blue Shark, Size: Medium
With my pitty pup, there's no such thing as an indestructible toy, but this has been the most impressive so far. It's lasted days vs the typical minutes his new toys survive. It has a squeaker, but no fluff, and this has been great. He's been trying to get at if for a long time with no success! He has tore thru the rubber portion, but there's still plenty of play left in this toy. The 3 different textures keep him interested vs the hard material toys he rejects or chews on them too long and I worry about tooth wear. Definitely buying more.
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Reviewed in the United States on June 7, 2026
★★★★★ 5
This has been a challenge for my extreme chewer! The toy is winning
Color: Green
Indestructible? Not for my pitbull. Except on this toy. She has slowly chewed some of the outer portion on the backside, but it still has a lot of gnawing time left. This is her go-to toy now. she carries it nearly everywhere. She loves it. I am amazed at how well it is holding up. Within minutes, hours or a few days, the other "indestructible" toys I've purchased, never survived as described.
This is a heavy toy! Not for the weak! I cannot stress enough the extreme chewer my dog is. Yet, I can see this toy lasting months. It won't be pretty then but it will most likely still be a favorite chew toy. I'm glad I found this and will be buying more.
FYI's... 1) Chewability-5 stars 2) Softness-1 star, it's solid but chewable, 3) Bounce-0 stars, No bounce, it's heavy!, Playability- 5 stars. My dog plays with it, lets it lay with her when resting. Nearly always within her reach. She sleeps with it! That's love.
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Reviewed in the United States on January 2, 2026
★★★★★ 5
My buddy has been chewing on this for an hour!
Color: Green, Color: Green
This dog toy has been a great addition to our dog’s toy collection! My dog can be a pretty aggressive chewer, so I’m always looking for toys that are durable and can actually hold up to daily play. This one has been surprisingly tough and has lasted much longer than many other toys we’ve tried.
The material feels strong and well made, and the design makes it easy for my dog to grip and chew. He loves carrying it around and tossing it during playtime. It’s also great for keeping him busy and helping reduce boredom, especially when he has extra energy to burn.
Another thing I like is the texture of the toy. It seems to help keep my dog engaged, and it’s sturdy enough that I don’t worry about it falling apart right away like some softer toys do. It’s been used for chewing, tugging, and even some fetch around the house, and it’s still holding up really well.
The only small drawback is that I wish it had a squeaker inside. My dog absolutely loves squeaky toys, and I think that would make it even more exciting and keep him interested for longer periods of time.
Overall though, this is a very solid and durable dog toy that’s perfect for dogs that love to chew. If you’re looking for something tough that can help keep your dog entertained and reduce boredom, this is definitely worth try
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Reviewed in the United States on March 12, 2026