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Description
Mouse YWHAe 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 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. 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 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 utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotinylated detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against Tyrosine 3/Tryptophan 5 Monooxygenase Activation Protein Epsilon (YWHAe). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of Tyrosine 3/Tryptophan 5 Monooxygenase Activation Protein Epsilon (YWHAe) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Tyrosine 3/Tryptophan 5 Monooxygenase Activation Protein Epsilon ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | 14-3-3 protein epsilon (YWHAE) is a protein encoded by the YWHAE gene. This gene product belongs to the 14-3-3 protein family, which mediates signal transduction by binding to phospholipid-containing proteins. This highly conserved protein family is found in both plants and mammals, and this protein is 100% identical to its mouse homolog. It interacts with CDC25 phosphatase, RAF1, and IRS1 proteins, suggesting a role in various biochemical activities related to signal transduction, such as cell division and regulation of insulin sensitivity. It is also thought to be involved in the pathogenesis of small cell lung cancer. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.2 ★★★★★
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Product Reviews
★★★★★ 5
Wide feet friendly
Size: 8 Toddler, Color: Tan River Gem
My son has always had extra wide feet and these sandals fit him well. We're limited on what footwear we can buy him and I'm glad I can add these to the list. He hasn't had an issue with these almost causing blisters like other sandals (Teva) we purchased last year. These are lightweight and stylish. I find these perfect for the pool and beach because they dry fast. They're cute enough for daily wear also. I will continue sizing up in these until they no longer have his size.
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Reviewed in the United States on September 11, 2025
★★★★★ 4
Great for summer and water but back strap was too big
Size: 8 Toddler, Color: Tan River Gem, Size: 8 Toddler, Color: Tan River Gem
These sandals were okay for my toddler. The sizing was right and the material is great for getting wet. They’re easy to get on and off and they can go with any outfit. The biggest con was that the strap behind the heel is a little too big so that my son’s heel isn’t secured. Every opportunity he got to take them off he would - in the car, on the lawn, at the beach etc. You can tell they weren’t his favorite sandal to wear and were slightly uncomfortable for him. The two Velcro straps on top were great and easy to adjust to his foot. Overall, they get the job done but I probably wouldn’t rebuy them.
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Reviewed in the United States on June 5, 2026
★★★★★ 5
Great shoes
Size: 10 Toddler, Color: Tan River Gem
Fantastic sandals! Easy for him to put on and don’t rub his feet even when wet. He loves to wear them anywhere, not just the water and they look great with any outfit. They seem to hold up well and have good traction. I wish they made these in bigger sizes! Definitely worth the money.
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Reviewed in the United States on June 13, 2025
★★★★★ 4
Good item
Size: 8 Toddler, Color: Blue River Gem, Size: 8 Toddler, Color: Blue River Gem
I got these Robeez toddler sport sandals for my little one and honestly, they’ve been pretty great overall. The first thing I noticed is how lightweight they are—they don’t seem bulky or heavy at all, which is perfect because my toddler is constantly on the move. They run around, climb, and play hard, and these sandals have held up really well so far. They feel durable enough to handle everyday wear without looking worn out after just a few uses.
Another big plus for me is how easy they are to put on and take off. If you have a toddler, you already know how much of a struggle shoes can be sometimes, so anything that saves time is a win. These slip on pretty easily, which makes getting out the door a lot less stressful.
That said, the sizing was a little off for us. I ordered my child’s usual size, but they do seem to run a bit big. There’s more room than I expected, so they’re not as snug of a fit right now. It’s not a huge issue since they can still wear them, but I probably could have sized down for a better fit.
Overall, I still really like these sandals. They’re comfortable, practical, and hold up well for everyday use. Just keep the sizing in mind when ordering!
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Reviewed in the United States on April 20, 2026
★★★★★ 5
Perfect for little ones on the move!
Size: 6 Toddler, Color: Blue River Gem
So far, the best pair my baby has. These are easy to out on and take off. They are easy to clean, and have been durable.
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Reviewed in the United States on October 5, 2025