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Description
Mouse Nestin ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant and centrifuge at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. Cell Supernatant: 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., 1 g of tissue sample to 9 mL 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 the tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and the supernatant can be assayed. Preparation before testing: 1. Please remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Preparation of gradient standard working solution: Add 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, and then gently mix (concentration is 10ng/mL). Then dilute to the following concentrations: 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.3125ng/mL, 0.15625ng/mL, and 0ng/mL. Serial dilution method: Take 7 EP tubes and add 500uL of universal diluent to each tube. 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 step to mix thoroughly. 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 Nestin capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Nestin in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Nestin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Nestin is a protein encoded by the NES gene. Nestin is a type VI intermediate filament (IF) protein. These IF proteins are primarily expressed in neurons and are involved in radial axonal growth. Seven genes encode the heavy neurofilament (NF-H), medium neurofilament (NF-M), and light neurofilament (NF-L) proteins: nestin and α-internexin in neurons, synemin α and desmuslin/synemin β (two alternative transcripts of the DMN gene) in muscle cells, and syncoilin (also in muscle cells). Most members of this group prefer to associate in tissues as heteropolymers. Nestin forms homodimers and homotetramers in vitro but does not form IFs on its own. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein internexin to form heteromeric coiled molecules. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, cell supernatant, tissue homogenate, etc. |
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4.3 ★★★★★
Based on 6 reviews
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Product Reviews
★★★★★ 5
Durable, Engaging Toy That Actually Lasts
Color: Brown, Pattern Name: Tree stump 2 Pack
I’m really impressed with the ClariVora 2 Pack Dog Toys for aggressive chewers. My dog is a strong chewer and usually destroys toys quickly, but these have held up really well. The material is durable and tough without being too hard, which gives me peace of mind.
I especially love that they can be used as frozen treat toys. Filling them and freezing them keeps my dog busy for a long time and really helps with boredom and anxiety. They’re perfect for extending playtime and keeping dogs mentally engaged.
These toys are a great size for medium to large dogs, easy to clean, and have already become a favorite in our house. If you have a power chewer and want something interactive and long-lasting, I definitely recommend these.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 30, 2025
★★★★★ 5
Small but aggressive chewers love these
Color: Brown, Pattern Name: Tree stump 2 Pack
I have a Jack Russell and a chihuahua/jack mix (I know, I must have done something really bad in a former life and this is my punishment). They’re both aggressive chewers for their size. These hold up well and they love them. The silicon molds are really easy to use and clean. I spread a little peanut butter around the sides and then fill with wet food, pumpkin, Greek yogurt etc. Once frozen they pop out easy. Keep the kids busy for a solid 20mins. Just don’t over-tighten when you close them!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 2, 2026
★★★★★ 5
Amazing durable product for your fur babies!
Color: Brown, Pattern Name: Tree stump 2 Pack, Color: Brown, Pattern Name: Tree stump 2 Pack
We love this product! I have two aggressive chewers, one being a Staffordshire bull terrier, and the other great Dane mix puppy who is eight months old. They both love to chew and can be mischievous or anxiety ridden when they are bored and if they aren’t entertained with something. My Staffordshire bullterrier is a very aggressive chewer and we’ve had a hard time finding durable toys for her. This product is amazing. It keeps them entertained for at least an hour with the food that you can place inside. The molds are great and it’s really nice that I can make homemade dog treats to put in the toy. I use these toys every day, multiple times a day, and they’re holding up amazing. I love that the company includes the molds for the treats as well as a little key to open up the back of the toy to put the food in. This is a really good product. I have spent hundreds of dollars on toys and this one is worth the cost. I’ve tried bully box, Kong products, anything you can think of that says durability chewing, however this is really really good. I 100% recommend this product and I think it’s very fair pricing. Your fur babies will get a lot of playtime with this!!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 20, 2025
★★★★★ 4
Good puppy toy
Color: Brown, Pattern Name: Tree stump 1 Pack
Keeps the pup busy. He is not able to bite through it, it is very durable. Only gave 4 stars because it only takes him about 15 minutes to go through it and hes onto the next thing. Love that he is not able to destroy it though like other toys.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 17, 2026
★★★★★ 5
Great toy
Color: Brown, Pattern Name: Tree stump 2 Pack
Love this for my dogs! Durable and easy to use. They love it too.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 28, 2026
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