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Description
Rat C4 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 specimens using EDTA or heparin as anticoagulants and centrifuge them at 1000×g for 15 minutes at 2-8℃ within 30 minutes of collection. The supernatant can be tested or stored at -20℃ or -80℃, but repeated freezing and thawing should be avoided. 3. 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 400 ng/mL). Then dilute to the following concentrations: 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 400 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 200 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 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 4 (C4) 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 4 (C4) 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 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Complement protein 4 (C4) is a protein involved in the complex complement system, derived from the human leukocyte antigen (HLA) system. It plays a number of key roles in immunity, tolerance, and autoimmunity, along with numerous other components. Furthermore, it is a key element connecting the entire systemic recognition pathway, initiated by the antibody-antigen (Ab-Ag) complex, with other effector proteins of the innate immune response. For example, severe complement system dysfunction can lead to fatal illnesses and infections. However, C4 protein is derived from a simple, bifocal allelic model, the C4A-C4B gene, allowing for abundant variation in the levels of its respective proteins within the population. The C4A-C4B genetic model, originally defined in the context of the Chido/Rodgers blood group system, is currently under investigation for its possible role in the risk and development of schizophrenia. | |||||||||||||||||||||||||||||||||
| 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 | 6.25-400 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, and other biological fluids |
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4.8 ★★★★★
Based on 188 reviews
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Product Reviews
★★★★★ 5
Beta, Alpha, Omega oh my!
Format: Kindle
Omegas are precious and given to Alphas & their packs... but the Betas want in too. To this end, the Beta government is rolling out its trial of assigning a Beta to each Alpha-Omega pack. But forcing a Beta into a pack where they are not wanted will not end well... Of course, no one expected the Omega to fall for the assigned Beta. Great read and cliffhanger
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 15, 2025
★★★★★ 3
A familiar story, just with…..less.
Format: Kindle
So, as other reviewers make clear, this is very similar to Pack Darling and The Beta. It’s much closer aligned with The Beta, in plot and maybe more like Pack Darling with characters.
That being said, I don’t hate this…..but it wasn’t great either. It’s both books mentioned but just….less. Less angst, less emotion, less feeling. The plot feels very half fleshed out, and the “bad guy” feels underwhelming. I didn’t really feel any real emotions from and of the male leads, except maybe Oliver. The others fell sorta flat for me. And Mika makes herself out to be this big bad ass straight outta training and then we never see it from here again with the one fitting room incident as the exception.
SPOILER:
The whole, “Oh, I’m actually probably an Omega, but I don’t wanna be but I do actually wanna be but no one can ever know my secret that I do nothing to hide “ thing fell so flat. She never commutes to believing she was secretly an omega, but also mentions her “secret” a lot. It just felt so manufactured.
I’m intrigued enough to read part 2 and see how the author closes everything out, but this is not one I’ll recommend or ever come back to.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 13, 2024
★★★★★ 5
Great book
Format: Kindle
I absolutely feel in love with all 4 characters!!! The bedroom scenes were 🌋🌡🔥🔥🔥. I couldn't put this book down!!! I'm hooked for the whole series Book 2 here I come!!!!! Its a fun easy book and story to read!!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 4, 2024
★★★★★ 4
Fun with a late blooming omega
Format: Kindle
I like this book. The story is fun, cute, and sexy. There's just a little drama, some excellent, steamy scenes, and a fairly good relationship building storyline. I especially like how all the main characters are a bit older than the usual 20 somethings I tend to see in this kind of book.
Having said that, I wish there were more descriptions of the places, as well as the food in the fancy restaurant. I enjoyed the cocktails at the club, so I missed that kind of detail when Gray took Madison on a dinner date. I also wish there had been more interaction between Lucas and Madison, and Lucas and Rian. It felt a bit lopsided, with a focus on Rian, Madison, and Gray.
I wish it had been proofread - there are a lot of typos, but nothing too distracting.
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Reviewed in the United States on September 12, 2022
★★★★★ 3
Madison Deserved Better
Format: Kindle
Madison was a beta...except she wasn't any longer. She was a late presenting Omega. And she was struggling. She was tall and thin, not tiny and curvy. She was opinionated. She was everything an Omega was not. After suffering through her first heat, her friends took her to Ardor, a club where Omegas came to safely find Alphas. She's not expecting much but then she connects with a sexy beta. And when she meets his Alphas, they set her body on fire. Maybe, she's found her no-strings-attached heat pack. Maybe, she's found something more.
I could not connect with the characters in this book, so their story never resonated with me. And there was no love story; there was sex. Grey made it clear from the beginning that he had a true love and it was his beta boy, Rian. He went so far as to reassure Rian “Say the word, I’ll never touch her again. Lucas can put the babies in her. I only need you, beta boy”. So, Madison was there for babies, no emotions needed. Nice. No, thank you. I want the Omega to be the center of their world, not an incubator. Lucas and Rian weren't any better. After her heat, they let her leave. Not one of them made her feel valued. No one gave her a reason to stay or even offered a cuddle. And the sex didn't even come across as mind-blowing. Madison deserved better.
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Reviewed in the United States on March 11, 2025
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