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Description
Mouse DEFa6 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: Serum, plasma, 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
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: Serum, plasma, 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 freeze-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 freeze-thaw cycles 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 1000×g for 20 minutes, and collect the supernatant for analysis. Preparation for the Assay: 1. Remove the reagent kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 1000 pg/mL). Then dilute to the following concentrations: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 1000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500 pg/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 Defensin Alpha 6/Paneth Cell Specific (DEFa6) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of Defensin Alpha 6/Paneth Cell Specific (DEFa6) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Defensin Alpha 6; Paneth Cell Specific ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Defensin α6, Paneth cell (DEFA6) is a protein encoded by the DEFA6 gene. DEFA6 is expressed in Paneth cells of the ileum. α-defensins are a family of microbicidal and cytotoxic peptides that protect the host against bacteria and viruses. However, DEFA6 provides protection against invading enteric bacterial pathogens by self-assembling into cellulose and nanomembranes that surround and entangle bacteria. Several α-defensin genes, including DEFA6, are clustered on chromosome 8. | |||||||||||||||||||||||||||||||||
| 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 | 15.6-1000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate and other biological fluids |
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4.6 ★★★★★
Based on 1884 reviews
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Product Reviews
★★★★★ 4
Strong and Great for Treats, but Heavier Than Expected
Style: Gorilla, Size: Small (Pack of 1), Style: Gorilla, Size: Small (Pack of 1)
This is a very durable and well-made chew toy with good overall quality.
The material is thick rubber and holds up well to chewing. It also works very well for holding treats, which stay in place securely.
The main drawback is the weight. My dog is about 30 pounds, and even though this is a smaller version, it is still fairly heavy. She tends to use it mainly to get the treats out and then loses interest afterward.
What I like:
Very durable material
Works great for holding treats
Good bounce and quality construction
Cute design
Things to be aware of:
Heavier than expected
Some treats may be difficult to insert
Dogs may lose interest once treats are gone
Overall:
This is a solid treat-based toy that is built well, but the weight may limit how much some dogs interact with it beyond treat use.
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Reviewed in the United States on May 23, 2026
★★★★★ 5
Extremely tough!
Style: Gorilla, Size: Large (Pack of 1)
These are seriously so much better (and cheaper) than any other supposedly "strong/durable" type toy like this. We have 70lb very strong chewers and they chew through everything. Can not have stuffies. They do eventually chew through but it seriously takes awhile. Especially in comparison to other brands. I will say it says "recommended for dogs under 35lbs" but I also work for houses where I recommended these for their dogs which were smaller and they wouldn't chew on because too tough. But they also accidentally bought the large which is what I buy my big dogs. So the large is GREAT for big dogs. If you have a dog under 35lbs I'd get the size small. Also .... says you can stuff treats. You really can't. Which my dogs don't need. They just love to chew on. I call it their bubble gum because when they FINALLY get a chunk off(which again takes awhile.... as in over a week) they will fling the pieces into the air and chew on like a piece of bubble gum lol... they love it! I don't notice a difference in breath or anything but I don't use the toothpaste either. I just bought to see if tough and they are!! And again price is clearly so much better. I hate spending $20+ on a toy they destroy in a day.
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Reviewed in the United States on December 30, 2023
★★★★★ 3
Beware if your dog likes to chew
Style: Gorilla, Size: Large (Pack of 1)
It’s super cute and my dog loved it, but it didn’t last very long. My dog started peeling off pieces of it and eating it within three hours of play.
If you have a dog that takes it easy on the toys I would recommend this one. Otherwise I recommend passing because there’s no way it’s safe for them to be swallowing these pieces.
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Reviewed in the United States on March 26, 2026
★★★★★ 5
Lasted pretty long for my aggressive chewer
Style: Gorilla, Size: Large (Pack of 1)
Right away I was excited with this product because of the smell, texture and thickness. Smells minty fresh which does help with breath smell. The texture is great, had smooth areas and rigid areas all while being soft and durable. The product is pretty thick, which I needed for my Pit-Bull. She doesn’t chew much but, when she decides to she is 110% in and dedicated to destroying that item. She chews through most things in less than a day. This product however lasted years before I started to notice damage. It is one of her favorite toys. She liked it so much I bought two, one for the house and one for the car for road trips. It’s the perfect toy to keep a chewer distracted inside a car, along side a deer antler, which also helps with dental hygiene. She gets dental hygiene compliments every trip to the Vet. We use multiple different things to help with her teeth and breathe but, I think this toy contributes the most to her healthy teeth. I call it a toy because she plays with it all the time. Definitely will keep buying long as they keep making it. This product is a must for all dogs, it’s almost perfect for my Pit-Bull
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Reviewed in the United States on February 24, 2021
★★★★★ 5
Currently surviving a SUPER CHEWER!
Style: Gorilla, Size: Small (Pack of 1), Style: Gorilla, Size: Small (Pack of 1)
Welcomed a pomeranian/terrier mix to the family recently. Although he is small he is very capable of tearing things apart! He has destroyed many, many types and brands of toys. When he grew tired of destroying those he turned to objects in my house, walls, furniture, railing etc! I've tried different brands and textures and this is one of only 2 brands he hasn't outright destroyed! He's had this toy for almost a month now and it has come out of this time almost completely unscathed! You'll notice there's a little damage to one of the hands and top of the head but hes managed to destroy toys in an hour or less, so I consider this a big win and will absolutely replace with the same product when the time comes! There's a hole, I think for treats. I haven't used that. It also has a smell, almost like baby powder. In any event, absolutely worth the price which I found to be great to begin with. My dog loves it, and me and my house are soo glad he's chewing on this! If you have a super chewer (I'd never heard of that) I strongly suggest this toy. They also have larger ones for bigger breeds!
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Reviewed in the United States on September 6, 2025