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Description
Rat IL12A 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 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 100 pg/mL). Then dilute to the following concentrations: 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL, 6.25 pg/mL, 3.125 pg/mL, 1.5625 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 100pg/mL standard working solution into the first EP tube and mix thoroughly to make a 50pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used 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 competitive enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled antibodies, and HRP enzyme conjugates are added sequentially to microwells pre-coated with the universal species Interleukin 12A (IL12A) antigen. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to the final yellow under the action of acid. The depth of the color is negatively correlated with the universal species Interleukin 12A (IL12A) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Interleukin 12A ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Interleukin-12A (IL-12A) is a protein encoded by the IL12A gene. This gene encodes a subunit of the cytokine interleukin-12 (IL-12), which has a wide range of biological activities on T cells and natural killer cells. This cytokine is a disulfide-linked heterodimer consisting of a 35 kDa subunit and a 40 kDa subunit encoded by this gene, the latter being a member of the cytokine receptor family. This cytokine is required for T cell-dependent induction of interferon gamma (INF-γ) and is important for the differentiation of both Th1 and Th2 cells. Lymphocyte responses to this cytokine are mediated by the transcriptional activator STAT4. Nitric oxide synthase 2A (NOS2A/NOS2) has been found to be required for signaling by this cytokine in innate immunity. | |||||||||||||||||||||||||||||||||
| 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 | 1.56-100pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.6 ★★★★★
Based on 1659 reviews
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Product Reviews
★★★★★ 5
Excellent pee pad tray has "legs" to keep feet off of the pad
Size: 18"X24", Size: 18"X24"
My 10 week old Pekingese learned quickly to use it, and Pekingese are a very stubborn breed. We started training her by removing the grid because she was used to paper. It snaps out easily. We used pee pads with sticky tabs to attach the pad, then snapped the "frame" in place without the grid. Within a few days, she had figured it out, so we snapped the grid back in so her feet never touch the used pad. No more wet feet. I bought the medium size that is aqua blue.
For those that ask if it will fit their crate...measure your crate! My large crate is 42" x 23" and the tray fits two ways as seen in the pictures. My small crate is 23" x17" and the tray does NOT fit. I wish the company made a 10" x 14" tray for traveling. (pink???)
Start using it when you have a couple of days to give your puppy 100% of your attention. 100% attention!!
If she/he does not use it, then it is your fault for not paying attention. My puppy would go every 15 minutes at first! Put a WHITE plastic shower curtain liner on your floor so you can see if she misses it. The liner wipes off easily with Clorox wipes or goes in the washing machine. For those puppies who stand on the edge of the tray, but still miss it because their back legs are barely on it, help them walk forward and put plastic under the tray to catch any misses.
Now, at 4 months old, she seldom uses it, but it is available. Her pen is getting larger and larger. After she pees outside, we let her run around in in the living room for about an hour, then she goes back in the pen. Her attention span is short and she can't always make it back to her tray in the pen. My husband always brings her in the house after she has peed one time outside, but if I take her outside, I wait until she goes at least 3 times and there won't be accidents in the house. Sometimes, it takes more than 1 time to empty their bladder.
This pad tray is not like others on the market. Look closely at other grids, and you will see that other trays do not have the "legs" on the under side of the grid that keep the plastic grid from touching the used pad. My puppy tried to chew on the plastic tray, but she is always in our vision so we just say, "Ahhh!" and she leaves it alone. No problems with chewing. The Ahhh!" stops her every time, and we say it to stop her from doing anything wrong. Some people say their dog chews on it. Just like children, dogs need to be taught. Don't complain about how the pee pad or pee tray doesn't work. Teach your dog or your dog to not chew on it.
Like others have mentioned, my puppy likes sleeping on the tray at night, because it has air circulating under it, so it is cooler than her bed. (Not the fault of the product!) We can't break her of that habit without confusing her or removing the tray. She may stop using it if we teach her to get her off it. I have been putting frozen gel compresses wrapped in a towel in her play pen during the day, and as long as the compress stays cold, she will lay on it instead of the pad tray. Always watch, so a dog doesn't chew on the gel compresses.
I don't know why some people have said that urine runs out, because it is sealed. Maybe they are not changing the pads often enough. I started out changing them twice a day, then since my puppy started flea and tick medicine, she could start going outside. I changed the pad once a day then. Now, at 4 months old, she goes outside, but we put it in her large kennel at night. (The tray is too big to fit in her small dog kennel.) For the last three days and nights, she has not used it at all, but it is there just in case.
If your tray does not have rubber pads on the bottom, buy some or put the tray on a piece of rubber kitchen shelf liner. My tray doesn't slide. If your dog runs to it and jumps on quickly, it may slide, so put something under it.
Remember, your dog may not want to walk on it, but it is up to you to teach the dog to walk on it. They don't get a choice! You are in charge!
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Reviewed in the United States on September 17, 2020
★★★★★ 5
Hands down, best pee pad holder
Size: 18"X24"
I did so much research to find the right puppy pad holder. I would give this 10
stars if I could.
It’s softer plastic but not flimsy. The holes are comfortable for puppy feet. I see a lot
with metal grates and pictures of injured doggy feet. Our pups are small and they walk on it just fine. I used to go through 10 pee pads a day. Now one pad lasts all day. Pups can’t go wee wee and trail it all over the house. Since it’s the same pee pad, pups look for the smell and are motivated to pee pee in the same spot. Puppies used to wake up several times a night because they would pee, step in it, tear up the pee pad, make a mess. The night time drama has definitely decreased significantly.
Easy to lock, puppies can’t get into it. Easy to open to change out the pee pad. Comfortable to clean in a larger kitchen sink. Lightweight. Sometimes the doodie will go through the grates and sometimes it sits right on top, depending on the size of the doodie. Cleaning doodie stuck in the grate is never fun, but I use an old toothbrush and it works just fine.
Very happy with this item.
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Reviewed in the United States on January 29, 2026
★★★★★ 1
easily destructed by puppy that chews
Size: 24"X24"
disappointed in this purchase. It comes in 2 pieces, which are not exactly easy to fit together and cumbersome. This design flaw also makes it very easy for puppies who are prone to chewing to destroy. It last 1 day then i had to toss it
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Reviewed in the United States on January 29, 2026
★★★★★ 3
Great, for the most part
Size: 24"X24"
Love it overall and super glad I chose this one after looking at so many.
Pros:
- Perfect size for what I needed. Fits exactly in the space it was planned for
- Fits 22x22 pee pads nicely (which are easily accessible and cheaper than other sizes)
- the color is a brighter blie than I was expecting and shinier in person, which I like very much :)
Con:
I didn't realize that it's actually 4 pieces.
2 for the top, 2 for the bottom, and you have to put them together.
- I like the little weight distribution pieces they have so it keeps the top from sinking down onto the pee pad.
A few problems with that:
- I'm pretty great at putting together furniture, but for some reason one level connected fine and the other just would not go. I ended up using a flat head screwdriver to pry a piece out so it would go over the other. (If you buy it, you might understand what I mean better lol)
- with it coming in 4 pieces instead of two, it's less structurally sound and tends to bow in the middle. Even more so because they put both middles parallel instead if perpendicular.
- there are potty pads in there, but the seams create a more likely scenario of dripping onto my floor. Where I wouldn't have to worry about that with a solid piece.
- I may have to take the whole thing apart to clean between the individual pieces and this may cause it to be less secure over time due to taking apart and putting back together over time.
Great little thing overall and glad I got it :) serves it's purpose
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Reviewed in the United States on February 7, 2025
★★★★★ 5
Works Perfect!
Size: 24"X24"
Same scenario here as a lot of other reviewers... We've got a 10-week-old pup that used potty pads at first... But now is in the teething and let's tear everything up stage. It was starting to get dangerous even with other holders she was tearing up the pads completely and we were worried she was going to ingest them.
We looked at several different products, including the over twice the cost metal wolf brand. But I have to say, really glad we went with this one. It does take a minute to snap together properly, but once you do changing out pads is a breeze.
Once our girl (Mini Poodle) went on top of the greats and used the bathroom we gave her a big reward with lots of praise and a treat. She hasn't been able to tear it up at all. And I believe since she can smell her marking, she has been going back and using it about 7/10 times.
More recently, the success rate has been going up. Like I said, she hasn't been able to flip it over, break it apart etc. So we are incredibly happy with this purchase and has made our lives so much easier.
Once she gets bigger we will see how she handled it. And if she is able to break it apart. But I doubt it, there is a fairly satisfying click when you lock in both sides with the pad beneath.
We are using Frisco pads from Chewy large size and they fit pretty much perfectly. You have to fold the blue border slightly but it works great!
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Reviewed in the United States on November 3, 2024
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