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Description
Human zonulin ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 3. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. 4. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. 5. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 6. 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 uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP enzyme conjugate are sequentially added to microwells pre-coated with a zonulin capture antibody. After incubation and washing, the assay is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of zonulin in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human zonulin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Zonulin is a protein that regulates the permeability of tight junctions between cells lining the digestive tract. It was discovered in 2000 by Alessio Fasano and his team at the University of Maryland School of Medicine. As the mammalian analog of the Zonula occludens toxin secreted by the cholera pathogen Vibrio cholerae, it is thought to be involved in the pathogenesis of celiac disease and type 1 diabetes. Gliadin, a glycoprotein found in wheat, activates zonulin signaling, independently of genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.5 ★★★★★
Based on 25 reviews
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Product Reviews
★★★★★ 5
The Best Clean Beauty Facial Sun Screen!
This spring I tried four different clean beauty facial sunscreens. This is my favorite. First, it goes on with a light tint that rubs in, which moisturizers and becomes a primer for my bare minerals powder foundation makeup. Absolutely no flacking after applying the powder. (Many times if I am just out walking, playing pickleball or working in my garden I just wear this and skip the makeup) Second, the scent is so nice and light. Third, and actually most importantly is the rating on Yuka of excellent and that it works very well in the sun. I have worked hard to find only clean beauty products for my skin and I so appreciate of companies that provide them at an affordable price. I highly recommend this sunscreen.
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Reviewed in the United States on April 11, 2026
★★★★★ 5
Very thick but it's great otherwise as a light indoors sunscreen
I didn't mean to buy two, so definitely gonna return one. Two ounces, but for $12 that's pretty decent! Looks like this is mostly made for just the face and neck, so if you use it for your whole body then yeah, not worth the price. But just for the face? This will last me a long time. Most nice face sunscreens are like $20 for 1.5oz so $12 for 2oz is great.
I didn't pay enough attention and saw that it was SPF 30 once it got here. Which has me very confused because there's 24% zinc in it. That's almost the cap. Much higher SPF products have half that amount. It also has other plant extracts which are said to boost spf so I really don't get why this is so low.
I'm hoping the ppd at least is high, even though we have no way of really knowing with American sunscreens and even companies who do measure are greedy with that information and refuse to tell. So we are left guessing most of the time.
But, SPF is UVB rays. UV-burning. Ppd is UVA. UV-aging. Apparently American sunscreens have terrible UVA protection, or they just don't tell us at all. So the fact a few people in here were saying they were tanning through this sunscreen shows me that perhaps it's not very high, or that this is more of an incidental "I'm not going outside too much" sunscreen. Which, not surprising. SPF 30 lol.
Its very thick and moisturizing, so this seems like it would be the perfect winter sunscreen when the UV index is low and the air is dry. Its also difficult to find tinted sunscreens for very fair skin. And on the off chance there is one, it's usually either too dark anyway, or has an orange tint.
This one may be a sunscreen not best for darker skin types but man is the tint perfect for the palest of us! It has light coverage too. I mean my flaws aren't blurred or anything but my acne scars are a bit lighter colored.
And the great thing about tint, is it gives protection again VL/visible light. Only tinted sunscreens offer that extra bit of protection. I just wish the spf were higher! It also has a very light orangey smell. Also is water proof. So they say. It washed off a bit too easy for me to believe that though.
All in all it's a nice sunscreen. But I don't think I will re-purchase this once I run out in the warmer months, though I may consider it again once fall hits.
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Reviewed in the United States on August 8, 2024
★★★★★ 4
Clean ingredients, great price, but...
I want to love this product but I personally can't stand the oily texture and the smell. Maybe if you have super dry skin it will feel great on your skin
PROS: The ingredients are great! super clean, the percentage of zinc oxide is perfect and almost the highest % recommended by dermatologists, so it protects the skin really well, no white cast because it's slightly tinted (a little cast at the beginning then it fades away)
CONS (in my skin): it leaves my face super oily I have to use rice paper after I apply it to absorb the oiliness [sigh], I don't like the smell it bothers me a lot, it smells like old makeup.
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Reviewed in the United States on June 25, 2024
★★★★★ 5
Perfect Everyday Tinted Face Sunscreen
I am a long-time user of Think body sunscreen and tried this to replace my current department store tinted face sunscreen, which is being discontinued. I was amazed how well this performed, given the low price! It is literally 1/4 of the cost of my current face sunscreen and I am almost as happy with it! It was much lighter in tint than I was expecting, but blended easily and provided more "cosmetic" coverage than my current tinted face sunscreen, but without looking making me look made-up. My skin is light with reddish undertones, and it really helped to tone the red down as well, an unexpected bonus. That said, I typically use make up that is either the 2nd (or occasionally 3rd) from the fairest shade in the line, regardless of brand, and I am not sure how well this product would work on someone whose skin is even a little darker than mine, without being obvious. It is definitely too light for my sons, who have more olive-toned complexions. This product is highly moisturizing, and does take about 10 minutes to "set" before I feel comfortable applying powder to cover up the shine it leaves (a little more than a "glow"). Its efficacy is excellent--I really put it to the test outdoors and was pleased to see I had not to picked up any color from the sun when I washed it off. The other big plus is that it did not make me break out, and I have what my dermatologist calls, "reactive skin," which means anything from chemicals in face products to the hairdryer at the salon have been known to set it off. Finally, like their body sunscreen this face sunscreen gets a safety rating of "1" from the Environmental Working Group. I think I have found a winner!
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Reviewed in the United States on September 3, 2019
★★★★★ 5
Nice face sunscreen!
Great face sunscreen! It has high zinc and looks nice under makeup. I will continue to purchase. Not sure why it’s only a SPF of 30. It has 24% zinc which is higher. It’s a little thicker than some of the thinner ones I’ve used but I like that. I feel I’m getting good coverage
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Reviewed in the United States on April 2, 2026