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Description
Mouse 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 | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Forkhead Box Protein P3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Forkhead P3 (FOXP3), also known as scurfin, is a protein involved in immune system responses. As a member of the FOX protein family, FOXP3 plays a master regulatory role in the development and function of regulatory T cells (RTCs). TRCs typically downregulate immune responses. 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. In a TRC model system, the FOXP3 transcription factor occupies the promoters of genes involved in TRC function and may repress transcription of key genes upon T cell receptor stimulation. The FOXP3 gene contains 11 coding exons. Genomic sequence analysis has mapped the FOXP3 gene to the p arm of the X chromosome. | |||||||||||||||||||||||||||||||||
| 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 23 reviews
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Product Reviews
★★★★★ 5
Trust me - the few extra dollars is worth a shoe that actually stays on!
Size: 6 Little Kid, Color: Red/Blue/Lime
In an attempt to not spend lots on money on shoes for my toddler since they grow so fast, I ended up spending more money buying different "affordable" shoes and not a single one stayed on my little one's feet! The cost of these shoes is not outrageous but a little more than I wanted to spend on shoes that will be grown out of in a few months. Totally worth the few extra dollars though! Don't waste money and time trying to "save" money on shoes that don't work.
Also, quality of the shoe is great. Got the red pair and they are adorable! My little one likes to run and these are holding up great! They stay securely on his fit and have plenty of room in the toe area.
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Reviewed in the United States on April 22, 2024
★★★★★ 4
One month later - Pros & Cons
Size: 10 Little Kid, Color: Cobalt Blue, Size: 10 Little Kid, Color: Cobalt Blue
I purchased these shoes for my three year old son who is really hard on his shoes. I wanted shoes that were a good price value and that my son could put on and take off by himself, run around in, and that would stand up to 3 year old kid abuse. These shoes fit the bill, and for the price I would recommend them and I would buy them again, but they'd be even better if they were even more reinforced around the toes.
**This is NOT a paid or subsidized review. If you find this review helpful, please click "yes" - thank you!
PROS:
* Price - these are a well made, good looking shoe for a very fair price.
* Easy for my son to put on and take off, but do not fall off. Velcro is holding up well so far.
* The velcro tab that holds them on is big, so there's plenty of room for error in putting them on, and there's plenty of velcro to ensure a good hold.
* Nice looking - my son likes them which is a big deal when you're three.
* Stand up well to rough and tumble play
* Lightweight
* Good grip on the soles of the shoes.
* Fit is as expected, no discomfort, he wears them all day long without complaint.
CONS:
* As with all kids shoes, the toe area could be even more reinforced although he's worn them every day for a month and, as you can see in the pictures, they're not showing much wear.
* VERY lightweight - so if you're looking for a heavier, more protective shoe for any reason, this is not the pair for you.
Overall I'd highly recommend these shoes, and would purchase them again.
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Reviewed in the United States on May 5, 2016
★★★★★ 5
Easy 2-Velcro closure
Bought the first pair at Kohls, was a favorite easy velcro closure for our 2.5 yr old Grandaughter. Wanted pink and Amazon had these. Perfect fit one half size up. They run a little narrow and smaller but are lightweight and easy for a toddler to put their own shoes on! Her favorite.
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Reviewed in the United States on August 9, 2025
★★★★★ 5
Perfect for my toddler
These shoes have been great for my 2yo! Easy on and off, stylish and affordable.
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Reviewed in the United States on December 16, 2025
★★★★★ 5
run small.
Size: 7.5 Little Kid, Color: Peri
I was a fool and didn't size up, our daughter was only able to sue them for a minute. Should have sized up 2 sizes, I know better. A bit chunky too which I forgot about with these shoes, but fun color and easy to put on.
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Reviewed in the United States on October 4, 2025