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Description
Human EPHB3 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: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend 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. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freezing and thawing. 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, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with an Ephrin Type B Receptor 3 (EPHB3) 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 Ephrin Type B Receptor 3 (EPHB3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Ephrin Type B Receptor 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ephrin B receptor 3 is a protein encoded by the EPHB3 gene. Diseases associated with EPHB3 include intracranial syndrome and subdural edema. Pathways involved include Akt signaling and nervous system development. Gene Ontology annotations associated with this gene include transferase activity, transfer of phosphogroups, and protein tyrosine kinase activity. An important homologue of this gene is EPHB1. | |||||||||||||||||||||||||||||||||
| 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, cell culture supernatants, and other biological fluids |
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4.9 ★★★★★
Based on 947 reviews
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Product Reviews
★★★★★ 3
Squeaky balls for big dogs
Color: Squeaky-4 Pack, Size: Medium
I wanted a cheaper alternative to the Kong squeaky balls but unless you have big dogs that have powerful jaws, it is challenging for small and some medium dogs to squeak these. My dogs lost interest when they realized they couldn't squeak them. I will have to pass them off to a friend who has large dogs.
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Reviewed in the United States on May 20, 2026
★★★★★ 5
Moms dog here…reviewing cos it’s mine!
Color: Squeaky-4 Pack, Size: Medium
Moms corgo here. Reviewing the squeaky balls mom bought.
First off..they’re green. I don’t know what green exactly is cos I’m colorblind, so whatever that is it is and because it’s mine, I like it.
Secondly, they’re fluffy. It’s an annoying texture that I like to peel off, but these are tough and peeling them is a struggle.
Thirdly, the bounce. I don’t know where these balls think they’re going to think my mom is so repulsive, but they bounce just high enough to catch them and bring the escapee back to mom when I feel like they need to go back to her, otherwise, I take them to a safe spot to release into the wild…but mom always captures them and they always escape. I tell you, it’s a vicious cycle.
Fifthly…the squeak!! That rotten vulgar noise!! I want to unalive those things so bad!! Balls in the past were no match for my jaws of death, but these?? These are resilient and have quite the will to live! I have yet to destroy one!!
To wrap this up…Corgo approves and declares them mine. I like them. I think your doggo will like them too. But you can’t have mine..cos they’re mine.
*drops the corgo mic*
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Reviewed in the United States on May 14, 2024
★★★★★ 5
Not for aggressive chewers
Color: Non Squeaky-12 Pack, Size: Medium
You get a good amount of balls for the price. My dogs loved them but unfortunately they did not last long. That is not the company’s fault, my dogs are very toy aggressive and destroy almost every toy.
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Reviewed in the United States on December 31, 2025
★★★★★ 5
Our German Sheppard loves these
Size: Medium
These cost a bit more than tennis balls, but they are so much nicer and longer lasting. For starters, they stay cleaner than tennis balls because they’re smooth rubber. Dirt won’t build up on them and if anything does stick, like grass or soil, it falls off once the dog slobber dries. They’re also thick, so they don’t fall apart or blow out like a normal tennis ball does in our dog’s jaws after 30 seconds. Our GS chomps on these like crazy and the only damage they’ve suffered is a crack that developed from the edge of the hole, but the crack is growing very slowly and none of these balls have totally failed yet. The balls do whistle when thrown ant high speed and that may help a dog track and locate it, but I’m not sure. Our neighbors hear the whistling too so it’s far from silent. Lastly the orange ball is easy to locate out in our yard, but the dark blue practically disappears.
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Reviewed in the United States on April 6, 2025
★★★★★ 5
Great for smaller dogs
Size: Small
These two balls are perfect for the smaller mouthed dog that loves to play fetch.
These balls are not only super durable (lots of teeth biting), but float in the baby pool we use for our miniature dachshunds.
The value here is much better than you’d find anywhere else. The noise, if bitten hard enough, was “low” at best. Easy to spot/find if overthrown. Will definitely buy again once these are in bad repair; so far, so good-love these for my fur babies!
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Reviewed in the United States on October 8, 2024