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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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4.0 ★★★★★
Based on 389 reviews
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Product Reviews
★★★★★ 4
Durable!
Color: 1 PC-Beef-Red
Alright, here's a product review for those Tough Dog Toys:
Finally, a Toy That Survives My Monster! - Tough Dog Toys Review
As the owner of a determined (and I mean determined) large breed chewer, finding toys that last longer than five minutes has been a constant and often frustrating quest. I've gone through countless plush toys ripped to shreds, rubber toys with chunks missing, and even some "indestructible" toys that met their demise far too quickly. That's why I was both hopeful and skeptical when I came across the Tough Dog Toys, designed specifically for aggressive chewers and large breeds.
Let me tell you, these bone-shaped nylon toys are living up to their name. My power-chewing Labrador has been going at this thing for days now, and I'm genuinely impressed. Where other toys have succumbed to his relentless gnawing, the Tough Dog Toy has held its own. There are some minor teeth marks, as expected, but absolutely no significant damage, no pieces torn off, and no signs of imminent destruction. "Almost indestructible" might actually be an understatement!
The design is simple but effective. The solid nylon construction feels incredibly durable, and the bone shape is easy for my dog to grip and maneuver. It's also a good size for a large breed – substantial enough that he can really get a good chew going without it disappearing in his mouth.
What I appreciate most is the peace of mind this toy provides. I no longer have to constantly supervise playtime, fearing that he'll ingest pieces of a destroyed toy. This feels like a safe and long-lasting option for even the most enthusiastic chewers.
Pros:
* Truly durable – stands up to aggressive chewing from a large breed.
* Solid nylon construction feels virtually indestructible.
* Safe design with no small parts to break off.
* Good size and shape for large dogs to grip and enjoy.
* Provides long-lasting entertainment.
Cons:
* May show some teeth marks over time (though this hasn't compromised its integrity).
* It's a fairly hard material, so dogs who prefer softer toys might not be as interested.
Overall:
If you're at your wit's end trying to find a toy that can withstand your aggressive chewing large breed dog, I highly recommend giving the Tough Dog Toys a try. This bone toy has proven to be the most durable option we've encountered, offering excellent value for money and, most importantly, a safe and engaging chewing experience for your furry friend. Five out of five paws!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 16, 2026
★★★★★ 5
Very well made chew bone!
Color: 1 PC-Beef-Red
Great durable, very well made chew bone! Our dog usually tears up all his toys within a few minutes or sometimes hours after he gets them. He’s had this bone for a few days and it’s still in one piece. Except for the rubber that’s in the middle, he chewed up in a couple of days. I would definitely buy more chew toys made by this company! Well worth the money!
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Reviewed in the United States on May 30, 2026
★★★★★ 3
The rubber middle is not durable, it lasted less than 3 hours.
Color: 1 PC-Beef-Red
I bought this as I have bully/husky puppies (9 months old) and a pitt/doberman (9 years old). They are heavy chewers and destroy everything. I saw a video of this toy. The woman said that her dog chewed on it for a while (about 2 hours or so) she showed the ends visibly chewed, which I expect but it was holding up. The rubber middle seemed to hold up to. I received this yesterday. I had to take it away from them last night as they were chewing the rubber middle off. They had it maybe 3 hours. I didn't give it a lower star score as I will just cut the rubber off and give it back to them. So I was disappointed in the rubber middle not being more durable but the hard plastic it's made from is the strong material they need.
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Reviewed in the United States on May 16, 2026
★★★★★ 5
Bones
Color: 1 PC-Beef-Red
Yes bone 🍖 is very strong and durable MIA, Rocky and Rambo love them ..
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Reviewed in the United States on June 9, 2026
★★★★★ 4
Smaller than pictured but still a good choice for LG-XL dogs/power chewers!
Color: 3 PC-Beef-Red, Color: 3 PC-Beef-Red
Fantastic that these come in a 3 pack - I wish more chew toys did this! The base for these chew toys is pretty tough! The center & ends are hard enough to withstand both my bully mixes & my Irish wolfhound/Great Dane cross. They’re safe enough for even the largest power chewers! (With Supervision) my American Bully got the rubber spikes chewed off in under 20 minutes which is mildly disappointing but he’s a true powerhouse & master destroyer of all chews so I’m not that bent out of shape about it. I do wish they were as large as they appear in the ad pics but isn’t that the game we all play ordering offline anymore? 🤷🏻♀️
• Pro Tip: smear this toy in peanut butter (xylitol free!), wet food mixed into a slurry with water or bone broth + kibble/treats, yogurt, or even pumpkin puree and freeze for a few hours & relax while watching all the organic engagement your dog gets out of this toy!
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Reviewed in the United States on June 3, 2026
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