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Description
Human FOXP3 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 utilizes 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 Forkhead Box Protein P3 (FOXP3). 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 Forkhead Box Protein P3 (FOXP3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Forkhead Box Protein P3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | FOXP3 (forkhead box P3), also known as scurfin, is a protein involved in immune system responses. As a member of the FOX protein family, FOXP3 appears to play a master role in regulating pathways involved in the development and function of regulatory T cells. In cancer, excessive regulatory T cell activity can prevent the immune system from destroying cancer cells. In autoimmune diseases, insufficient regulatory T cell activity allows other autoimmune cells to attack the body's own tissues. While the precise mechanisms of control remain unknown, FOX proteins belong to the forkhead/winged-helix family of transcriptional regulators and are hypothesized to exert control during transcription through similar DNA-binding interactions. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.3 ★★★★★
Based on 1530 reviews
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Product Reviews
★★★★★ 5
My dog loves this toy!
Color: Bunny (Gray), Size: Large, Color: Bunny (Gray), Size: Large
High quality, big toy, perfect for a medium to large size dog. After a few months of serious chewing I had to buy another one, but well worth the money! Rocky loves it.
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Reviewed in the United States on June 11, 2026
★★★★★ 4
Great toy but if your dog is an aggressive chewer, please think twice.
Color: Crinkle Chicken (Brown), Size: Large
I received this on May 23rd and on June 8th, after playing with this thing every day, our recently acquired 36 lb dog finally ripped it open and got out all the plastic and the squeaky part. Lily Belle will miss you, chicken.
Lily loooooved this toy! She'd toss it in the air, chew on it, pull it with her teeth etc. but as much as she loved it, we won't replace it. It's just not durable enough for her.
If your dog is an aggressive chewer, you might want to try a different toy. I hate that this lasted just a smidge over two weeks because Lily seemed to truly enjoy this toy.
RIP, squeaky chicken!
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Reviewed in the United States on June 9, 2026
★★★★★ 5
Great Value Toy
Color: Crinkle Duck (Blue), Size: Large, Color: Crinkle Duck (Blue), Size: Large
This toy has been one of my dog’s absolute favorites. He gets excited every time he sees it and will carry it around the house, toss it in the air, and keep himself entertained for a long time. The shape and texture seem perfect for him, and it quickly became one of his go-to toys over everything else in his basket.
What I really like is that it holds up reasonably well for the price. It’s definitely not indestructible, and after a lot of play, it will sometimes start to rip—usually around the feet first since that’s where my dog tends to grab and chew the most. But honestly, considering how affordable it is, I don’t mind replacing it when needed. It’s inexpensive enough that rebuying feels worth it because he genuinely loves it that much.
I’d rather keep buying a toy he is obsessed with than spend more on something that just sits untouched. It’s fun, cute, and keeps him happy, which is what matters most. Overall, I’d definitely recommend it for dogs who love plush toys and playful chewing.
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Reviewed in the United States on April 28, 2026
★★★★★ 5
Best affordable squeaker toy
Color: Crinkle Chicken (Brown), Size: Large, Color: Crinkle Chicken (Brown), Size: Large
my dog is obsessed with this toy, this has became his new comfort toy and after having this for about four months the legs are falling off but it’s very durable. The squeaker also moved from the head of the chicken to the body of the chicken but that’s after it was played with every single day. I love that it has two squeakers and it’s the perfect toy for my dog.
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Reviewed in the United States on May 6, 2026
★★★★★ 5
My dogs favorite toy EVER
Color: Crinkle Chicken (Brown), Size: Large
I have a Belgian Malinois who loves any and every toy, but this one is definitely her favorite. She will walk around the house constantly carrying it in her mouth, which is honestly adorable to watch.
The squeakers only lasted a couple of months, but I don’t really consider that a downside—I’m kind of grateful they didn’t last forever given how much she plays with it. It still remains one of her most-loved toys even without them.
My only word of caution is that if you work from home, this toy makes a lot of crinkling noise. And I mean A LOT. It’s great for keeping the dog entertained, but it can definitely get a little frustrating for humans trying to focus or take calls.
Overall, it’s been a big hit in our house and easily one of her favorite toys.
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Reviewed in the United States on June 8, 2026
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