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Description
Human™ 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 freeze-thawed. Finally, centrifuge the homogenate at 5000 x g for 5-10 minutes, and remove the supernatant for testing. For other biological fluids: Centrifuge at 1000 x g for 20 minutes, and remove the supernatant for testing. Pre-Assay 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 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 a Thrombomodulin/Soluble™ capture antibody. After incubation and washing, the sample 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 Thrombomodulin/Soluble™ in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Thrombomodulin; Soluble ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Thrombomodulin (sTM) is an integral membrane protein expressed on the surface of endothelial cells that acts as a cofactor for thrombin. It reduces blood coagulation by converting thrombin from a prothrombin to an antithrombin. Thrombomodulin is also expressed on mesothelial cells, monocytes, and a subset of dendritic cells. Thrombomodulin is encoded by the THBD gene. The protein has a molecular mass of 74 kDa and consists of a single chain with six tandem repeats of an EGF-like structure, a serine/threonine-rich spacer, and a transmembrane domain. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates and other biological fluids |
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4.1 ★★★★★
Based on 18 reviews
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Product Reviews
★★★★★ 5
A foaming cleanser that doesn't dry out my skin!
Style: Tolerance Daily Foaming Facial Cleanser
I have very dry, reactive skin and initially looked past this face wash because it is foaming which is usually a no-go for my skin. I am pleasantly surprised with how well my skin tolerated this product. No dryness or irritation and my face felt clean. I also love that it is fragrance free because fragrances seem to really irritate my skin and who needs to smell their face wash? I am happy to say this is another great product from Avene!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 2, 2026
★★★★★ 5
Gentle cleanser for dry and sensitive skin.
Style: Tolerance Daily Foaming Facial Cleanser, Style: Tolerance Daily Foaming Facial Cleanser
I have dry and sensitive skin, and this cleanser has worked very well for me. It is very gentle on the skin and has not caused any irritation.
The texture is light and foamy, it spreads easily across the face and rinses off without leaving any residue. After using it, my skin feels clean, soft, and comfortable, without any tightness.
The size is quite large, which I find great because it lasts a long time with daily use.
Overall, a simple but very pleasant cleanser for sensitive skin.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 13, 2026
★★★★★ 5
Holy Grail Moisturizer for Rosacea
Style: Restorative Protective Cream-3.3 Fl Oz
In seeing so many negative reviews I felt compelled to weigh in as this is my favorite moisturizer. I have very dry, menopausal skin. Additionally, I have rosacea which seems to have been a nice gift from said menopause (you can only laugh, right?). It's very common for those of us in our 50's to have a compromised skin barrier and when an issue like rosacea is present, even more so. Because of all this my skin is very intolerant to most moisturizers and nearly all actives (over the last few years I've had to discontinue my retinols and chemical exfoliators). While ingredients like ALA's, petrolatum, mineral oil, parafin/bees wax, and lanolin are well tolerated and known to be quite soothing, they don't hydrate very well as they are more occlusive than moisturizing. Enter this product. Magically, it does both! It's marketed as a protective cream, which again implies occlusive, but it penetrates like no other. Gone is the tight, dry feeling that persisted even when I had a heavy layer of product on my skin (my previous moisturizer was Eucerin Original Healing Cream -- which still has its uses -- because it was the only thing I could put on my skin outside of squalane oil during a flare).
Now, no topical can cure rosacea. However, the right product can calm the skin, repair the damaged lipid barrier, and improve its appearance. This moisturizer does just that and it's become my holy grail. Since starting this I've also added in the Cicalfate Serum which has only aided in repairing my skin. Another Avene product that works great for me is the XeraCalm Balm which helps with itching. I use all three of these daily and they've really helped fix the secondary issues that come with rosacea.
As for the complaints about it being greasy or too heavy. I can see this not working for people with normal or oily skin (or perhaps only in moderation). But my skin loves the heavier weight and because one of the ways I soothe a flare is to keep a tiny mister bottle on hand and repeatedly spray my face so I have to guard against the drying effects of evaporation. These products are so lipid heavy that I can wet my face all day long without worrying about that, though I do reapply throughout the day.
As for the watery consistency issue, I haven't had that problem but it does caution on the packaging not to expose the product to temperatures below 41 degrees F (5 C). I suspect that if it gets too cold either because of the weather or because of whatever warehouse it sat in during transit it will separate. Maybe open the package and before squeezing some out look to see if it has split and then shake it up/message the tube.
So, if you have skin like mine don't hesitate to try this product. I'm leaving this absurdly long review for my struggling brothers and sisters out there with rosacea in hopes that it provides some relief! Happy moisturizing, internet strangers. ;)
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Reviewed in the United States on July 18, 2025
★★★★★ 4
Possible solution to seperation
Style: Restorative Protective Cream-1.3 Fl Oz
I love this product so much, but, like everyone else the water HEAVILY separated. A lot of water just shot out immediately and as i've used the cream it has felt more and more thick bc of that, shaking the tube or not.
POSSIBLE SOLUTION:
I actually think the company could salvage this problem by upgrading packaging to a Cosmetic JAR and then customers could take a toothpick and stir if separation has occurred by the time of purchase. I hope to try this with my next tube, just toothpaste squeezed everything out into a nice cosmetic jar and stir the water back into the product. This is such a simple solution that could really solve the product waste.
This refreshes my skin, doesn't break me out, and is lovely. I break out so easily, and being middle aged my skin really requires a dense help with moisture. <3
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Reviewed in the United States on April 22, 2026
★★★★★ 5
Ordered in spring/summer and it arrived separated too BUT...
Style: Restorative Protective Cream-1.3 Fl Oz
Ok, So I heard great things about this cream and decided to order. I ordered on May 28th and I live on the Gulf Coast of Texas and it is in the low 90s right now. I was worried mine would arrived separated like many others claimed to have gotten theirs, and sadly it did. Warm weather or not, blue water shot out of the tube the minute I squeezed it. Luckily, I read Mia P's review and submerged my tube in warm water (hot water actually straight out of my instant hot water faucet). I left it for about 5 minutes then shook it vigoroously. It seemed to help but was still a bit wonky so I repeated the process again and success! My tube is totally fine now and comes out in a thick, rich, emollient cream as it is supposed to. I will update my review after I use it more but so far I love the way it feels on the skin. It is very thick but spreads nicely and completely absorbs into the skin and leaves no greasy residue which is a big deal because I have oily skin. I purchased this to help with my Retin A uglies (I actually am using Musely with .05% tretinoin) and am hoping it helps with the redness and peeling I am having. I will update my review once I've used it for a while but wanted to post this review as I saw so many people complaining about the separating and just thought I'd mention that you can fix this issue if you have it (and there is a good likely hood that you will).
Update: I've been using this for a week now and I am absolutely in love with this cream! It is the most hydrating and healing cream I've ever used. Usually, thicker face creams like this would cause issues for me with acne or clogged pores as I do have oily skin that can break out if I use products with occlusive ingredients, but this one does not. In fact, my skin has never looked better! This is a must-have if you are using Retin-A/Tretinoin and going through the red/peely phase with your skin. The separation that occurred when I first received it has not been a problem and the cream is staying thick and free from any strange liquidy issues after I put it in hot water as mentioned in the first part of my review.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 28, 2023
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