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Description
Rat C3 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 2000 ng/mL). Then dilute to the following concentrations: 2000 ng/mL, 1000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 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 2000ng/mL standard working solution into the first EP tube and mix thoroughly to make a 1000ng/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 conjugate are sequentially added to microwells pre-coated with Complement Component 3 (C3) 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 Complement Component 3 (C3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Complement Component 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Complement protein 3, often referred to as C3, is an immune system protein encoded by a gene called C3. It plays a central role in the activation of the complement system. One form of C3 convertase, also known as C4b2a, is formed from isoforms of C4 and the activated form of C2. C4b2a catalyzes the cleavage of C3 protein into C3a and C3b and is produced during activation through both the classical and lectin pathways. C3a is a noncytotoxic cytokine and a precursor of several cytokines, such as ASP, while C3b is a pro-proliferative agent. Factor I cleaves C3b into C3c and C3d, the latter of which plays a role in enhancing B cell responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3 convertase, composed of activated C3 (C3b) and factor B (Bb). | |||||||||||||||||||||||||||||||||
| 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 | 31.25-2000ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.8 ★★★★★
Based on 29 reviews
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Product Reviews
★★★★★ 4
Not for gnawers
Size: 11.5 in, Color: Bear
I was hoping this would work for my teenage ACD and save my leather goods from being chewed on.
He gnawed on it quietly I was thrilled. But I had to take it from him within a few minutes. Gnawing apparently equals chewing pieces off.
Not made for chewers or gnawers.
It seemed to be well made, was soft, was actually a fairly good size and didn’t get gross when it got spitty. Just didn’t work out for Captain chaos destructor of all.
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Reviewed in the United States on May 4, 2026
★★★★★ 5
Durable & Leather. For Small-Medium Dogs
Size: 15 in, Color: Retriever, Size: 15 in, Color: Retriever
This is a great dog toy! My Pomeranian is a small guy at only 8lbs. but he sure knows how to chew and he does it as often as he can. I was throwing out toy after toy because he was tearing them to pieces after only a half hour or so. After putting my money in the garbage so many times, I decided to search for toys that would hold up to his destructive mouth. I found a lot that claimed they wouldn't fall apart but they were all more than I wanted to spend. I came across this toy and a rabbit shaped one just like this. I bought both months ago and both are still in tact after all this time. Sure the edges are a little frayed but other than that, no damage whatsoever! I'm not sure what dura fused means but that seemed promising to me when I was reading the description, also the fact that they're leather made them seem more durable to me. Both have squeakers inside and some sort of stuffing as well. I throw all of my dogs toys in the laundry or hand wash them once a week, except for these. When these toys get wet with my dogs saliva, they look like they're not meant to get wet. Even without washing, these seem fine. This toy is probably about 9" long or so if I had to guess. In my opinion, it'd be perfect for a medium sized dog. It's a tiny bit big for my little guy but he manages just fine! If you're looking for a durable toy that your small to medium dog can chew on constantly, look no further!
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Reviewed in the United States on February 3, 2020
★★★★★ 5
Always looking for less destructible dog toys
Size: 8 in, Color: Squirrel, Size: 8 in, Color: Squirrel
I am constantly searching for aggressive chewer dog toys. So many claim to be and one look and you know they haven’t met your dog!! Our golden retriever is so sweet until he gets his claws and teeth into a new toy. We nicknamed him the shredder, as he rips his new toys to pieces. I accidentally stumbled across this toy. We have never owned a leather dog toy. The squirrel is going to give Maverick a run for his money. The edges are double sewn closed. I am very impressed with the quality of this toy for $5.75 which includes the sales price and an additional 10% off coupon. Even without those two incentives the toy is less than $8 prime. The other shapes are similar or cheaper in price and now I see a 50% off 1 toy coupon when you buy 2. What a deal and the squirrel has a squeaker inside. What I like about the squirrel is that there’s less limbs or appendages for Maverick to shred off. The bunny has ears, the raccoon has hands, the buffalo has legs, the bone and retriever stick have a rope, the bear has a neck and head that could be chewed off, similar to the fox who has a tail and snout. The squirrel looks like it would be harder to destroy! The squeaker was hard to push at first but is easier now. Maverick has a black lab sister who loves squirrels, more so than she loves toys, and I found this toy searching for squirrel toys for her. It is offered in 8 other shapes. The squirrel measures almost 8” tall by 5” wide. It’s perfect for a medium or large size dog. Based on the construction of this squirrel dog toy it looks acceptable for an aggressive chewer and won’t break the bank. It’s very affordable. Always supervise! Updates to follow at Christmas:). BTW, the squirrel did not smell bad upon opening.
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Reviewed in the United States on December 3, 2024
★★★★★ 4
Puppy loves it. Well built and fun for her to play with.
Size: 15 in, Color: Retriever
My double doodle mix loved this toy. It is well made and she loves the different texture of the rope. Sadly she has already torn the rope apart and so now it’s just the leather part. This would be the perfect toy for non agressive chewers. Wish the rope lasted a little longer. SPOT is my new favorite company to get her chew toys from now. They’re well built and aren’t expensive like all the other dog toys out there.
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Reviewed in the United States on June 30, 2025
★★★★★ 2
Destroyed in under 10 minutes
Size: 9 in, Color: Large Bone
I bought 2 of these toys, one for my 9 year old great dane who isn't an aggressive chewer, and one for my 6 month old puppy who is average.
In less than 10 minutes both were destroyed and my dane managed to swallow a good chunk.
I wasn't surprised when they both had small holes on the ends and started pulling the stuffing out. The stuffing and squeakers were confiscated immediately, but I let them keep chewing. By the time I came back from throwing the insides away, both dogs had chewed the toys in half and my dane swallowed a chunk when he saw me coming. The puppy was going to do the same thing, but luckily we're working on his "drop" command so he was excited enough for the bigger payout and dropped it.
They didn't just come apart at the seams, the whole toy ripped in half making it too easy to swallow. Neither of them are aggressive cheers and the toys didn't hold up at all. A stuffed animal toy would have lasted longer (and has any time I've allowed it). 10 minutes of playtime is probably too generous , but I'm trying to be fair. If I hadn't been right there watching them, they both would have been able to swallow the whole thing in minutes. Definitely not safe for any amount of actual chewing.
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Reviewed in the United States on November 14, 2025