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Description
Rat IL-35 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: 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: 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. 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. 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. 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. 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. 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 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 1000pg/mL). Then dilute to the following concentrations: 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 1000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500pg/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 IL-35 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 IL-35 content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat IL-35 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Interleukin-35 (IL-35) is an anti-inflammatory cytokine of the IL-12 family. As a member of the IL-12 family, IL-35 is produced by a wide range of regulatory lymphocytes and plays a role in immunosuppression. IL-35 can inhibit the development of Th1 and Th17 cells by limiting early T cell proliferation. IL-35 is a dimeric protein composed of the IL-12α and IL-27β chains, encoded by two separate genes, IL12A and EBI3 (Epstein-Barr virus-induced gene 3), respectively. Its receptors consist of the IL-12Rβ2 (part of the IL-12R) and gp130 (part of the IL-27R) chains. In contrast to these two related interleukins, IL-35 can signal through only one of these chains. This has been demonstrated in vivo, as the absence of either receptor chain does not affect the effects of IL-35. On regulatory B cells, IL-35 signals through the IL-12Rβ2 and IL-27Rα subunits. | |||||||||||||||||||||||||||||||||
| 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 | 15.6-1000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.2 ★★★★★
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Product Reviews
★★★★★ 5
On the fence? This is the sheepskin to get!
Size: 2' x 3' (Natural), Color: Wolf Tipped
I've been reading all the reviews for sheepskins on AMZ and felt this company was the one to go for and WOW did they deliver! The sheepskin came well wrapped but not vacuum packed and is even prettier in person than the pictures. It is super soft, fluffy and dense and mine measured 26" x 40". I absolutely love the wolf tipped colors: ivory, dark grey and a slight brownish hue. There is a slight odor but I don't find it offensive. It stayed put on my leather couch and sitting on it during a Netflix movie was heavenly warm and comfortable. I did not observe any shedding. Will definitely buy another wolf tipped one, once it becomes available as it is worth the money.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on January 14, 2026
★★★★★ 5
Gorgeous 😍
Size: 2' x 6' (Natural), Color: Champagne, Size: 2' x 6' (Natural), Color: Champagne
Absolutely beautiful!!! It is thick and luxurious, super soft and cozy. It’s really high quality and it shows. You can tell it’s natural without the burn test even from its cute little curls. All brushed (your pet brush is perfect 😉) out it looks stunning and fluffy. I got the champagne color and I love it! I don’t see any seams on the double pelt and it is substantial, not at all like it will disintegrate in a few years, but rather will become a much loved family heirloom. Well worth the investment! It is absolutely heavenly to perform yoga on by the fire 😌I love taking my slippers off and feeling the soft fur. I don’t want to get off it lol. I understand all those other customer’s kids and cats 🤣😂🤣
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Reviewed in the United States on November 26, 2023
★★★★★ 5
This is the one you want...
Size: 2' x 6' (Natural), Color: Natural (Yellow Undertones), Size: 2' x 6' (Natural), Color: Natural (Yellow Undertones)
I bought a ‘Genuine Sheepskin Rug Double Pelt Natural White Fur 2x6’ from Amazon in 2011 for $112.49, and recently decided to recommission it in my new living room. It looked so nice that I decided to add a second rug, and was pleased to find what Amazon claimed to be the same rug still available for only $83.78. Now I’m not one of those silly folks who expects the color and/or size of a natural product like a sheepskin rug to be perfectly predictable, and my living room is large enough that I didn’t need the two rugs to match perfectly, but when the new rug arrived, I couldn’t have been more disappointed. It was about 40% smaller than my older rug, was way, way off from anything that could be called white, and looked cheap, old, and tattered. I tried to comb it out a bit to see if I could make it look presentable, but it was a lost cause.
Amazon to the rescue. I returned the rug to Amazon for a full refund the next morning, and the transaction couldn’t have been simpler. And I immediately ordered the ‘Genuine Sheepskin Rug Double Pelt Natural White Fur 2x6’ shown on this page from Super Area Rugs for $119.00. What a difference! These rugs are large, plush, and super soft. The quality of these rugs is obvious, much, much nicer that either my old sheepskin rug or the ‘bargain’ rug I returned to Amazon. I was so impressed with the quality of this rug that I immediately ordered a second, which arrived this morning (it matches the other rug perfectly). My older sheepskin has been reassigned to another room because, well, it’s not in the same league as my two new rugs, which look terrific together.
As is so often the case, you get what you pay for. So if you’re looking for a top quality sheepskin rug, this is the one you want.
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Reviewed in the United States on December 23, 2015
★★★★★ 4
Very nice fleeces
This is a clean, soft, well groomed fleece intended for use on a 6 foot long sofa, so bought 2. They moved too much as a result of shifting bodies on the leather couch, so will get a longer version, sewn bottom to bottom, and used each of these fleeces on both armrests, instead.
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Reviewed in the United States on August 15, 2025
★★★★★ 5
Excellent Product!!! Will purchase again!
Size: 2' x 3' (Natural), Color: Wolf Tipped
Instead of ordering a cover for reclining loveseats, I chose to drape one of these over the headrest on either end of our loveseat. Not only do they protect the leather, they also help to elevate our decor. The rugs were received in a very timely manner. They are super soft, & warming as we move into the winter months. We've had family & friends comment on how great they look. 2 of them have already ordered, & added them to their sofa/loveseat/recliner decor. Have an issue of them slipping, add some velcro in between the crook of the cushion & the back of the furniture piece. It doesn't show & will help to keep the rug in place; if your busy body.!
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Reviewed in the United States on December 10, 2025