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Description
Rat IL-17 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 300pg/mL). Then dilute to the following concentrations: 300pg/mL, 150pg/mL, 75pg/mL, 37.5pg/mL, 18.75pg/mL, 9.375pg/mL, 4.6875pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 300pg/mL standard working solution into the first EP tube and mix thoroughly to make a 150pg/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 Interleukin 17 (IL-17) 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 Interleukin 17 (IL-17) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Interleukin 17 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Interleukin-17 (IL-17), also known as CTLA-8, is a polymorphic cytokine expressed by T cells that shares high homology with the protein encoded by the ORF13 gene of the herpes simplex virus Saimiri. cDNA clones encoding IL-17 have been isolated from activated rat, mouse, and human T cells. The human IL-17 cDNA encodes a 155-amino acid (aa) precursor protein with a 19-aa signal peptide, which is cleaved to produce a 136-aa mature IL-17 containing a potential N-linked glycosylation site. Both recombinant and native IL-17 have been shown to exist as disulfide-linked homogenates. | |||||||||||||||||||||||||||||||||
| 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 | 4.68-300 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.2 ★★★★★
Based on 804 reviews
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Product Reviews
★★★★★ 5
OUTSTANDING
Format: Kindle
This was very, very good. The world is vast and characters are complex. There is a good plot with a whole lot going on. This is well written. Good twists and turns and some heart breaking moments. You will love these characters, they have heart and loyalty. I am hoping that there will be several more books. We've yet to see anything from the Sea Court but only a mention of them here and there. The Wood Court was given a quick couple of scenes, and only as far as some warriors, we've yet to enter their court and the Shadow Court, I'm not sure if they will be a force for good or bad, but they definitely will play a much bigger role moving forward. This is primarily the Ice and Air Courts. Told in multiple views, which I loved, it gives you a chance to see things from different eyes. There's alot of political maneuvering and deception. I loved it and will pick up the next book as it becomes available. If you like The Fae and the courts, you should love this. I think the author has mucn in store for us.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 8, 2020
★★★★★ 4
Definitely worth the read!
Format: Kindle
After taking a deep breath and taking in that wicked twist of an ending, I have finally composed myself. My first thought when I started this book was that I love Reyna's character. I was intrigued by her connection with her familiar and the Ruin that is plaguing her land. It came as no surprise that she took her sister's place in an attempt to protect her. When they reach the Air Court everything slows down. This is where it was iffy for me. First of all, I like multiple POV's in books however 7 is a bit much. It starts to interrupt the story line. I felt like I was finally making progress connecting with one character, then it was switched to another person. I felt they all had necessary or pertinent information but not necessarily were they all POV worthy. The only other thing that annoyed me was that Reyna constantly was " trapped." She would rush off without thinking, only to need rescuing. She is brilliant in a fight, but she really doesn't think through anything. Lorcan is amazing. I know he might be on the "bad" list, but his background is so interesting. Eislyn(Reyna's sister) is really so sweet, but calculating. I enjoyed her and Thane's dialogue. The author did an amazing job with the imagery in this book. Everything was so detailed it was easy to fall into the scene. I love unexpected twists and while part of the ending I expected, I wasn't expecting how it took place. All in all, I found it very entertaining and I am very invested in continuing this series.
Favorite quotes:
"The truth may be twisted but never false."
"Who was she if she was not the enemy of the Air Court? What was her purpose of she no longer has that?"
"In a war-torn land, love was always a lie."
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 4, 2020
★★★★★ 5
Great Read!!! Great story!!!
Format: Kindle
The series is long, but Ms. Wolfhart does a fantastic job of weaving this tale while bringing so much to the characters. Surprises and plot twists along the way to keep you intrigued. There is some graphic sex, but is no way the focal point. Grammar was excellent (a rare find with a lot of self publishers) with only a few noted errors. I rarely give 4 stars, let alone 5.
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Reviewed in the United States on April 30, 2021
★★★★★ 3
Sadly, DNF
Format: Kindle
I read this thru KU. I LOVED the synopsis. And then I began reading... and it was a DNF at 68% after picking it up and putting it down several times because I really loved the main female character.
*****SPOILERS*****
Pros:
The world is unique, intriguing and fun. The primary female character is bad-a** but not a b*tech or a mary sue. The primary female has depth. I really want to know what happens to her even tho it's been weeks and I don't remember her name. The villains to the point I read are pretty good -- an ever present threat of mysterious and possibly many culprits.
Cons:
Way, way too many points of view. I stopped counting at 7. It's the prime reason why I don't care about most of the characters or remember their names even when I like them. There's just too many points of view so almost none of the characters have enough book space for the author to properly develop them. This literally killed the book for me. Actually it killed my desire to read. For weeks.
The main male is more villain than hero. He agreed to marry the main female then locks her up & eschews her for her sister, all while bad mouthing her as unfit to rule when he never spent any time with her getting to know her. He is actually unfit to rule as he is blind to the woes of his own kingdom and starts off a peace mission to secure a ceasefire through marriage by murdering an inn full of people in her country for no real reason. Plus, he constantly makes promises he does not keep. And it's gross of him to pine for the sister behind the main female's back.
*****
As much as I really wanted to see what happened to the main female character, it wasn't enough for me to keep trying to slog thru this book. There was a lot of potential here that just fell short.
Hence, 3 stars.
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Reviewed in the United States on June 19, 2021
★★★★★ 5
Fae Courts with High Intrigue
Format: Kindle
This book is one that just builds and builds and then surprises you to no end. You may think you know the villains and then you are jolted in another direction. Princess Reyna is a real gem, strong of character, a fierce fighter, and loyal to her family and kingdom. Just when you think she and Lorcan, well you know, the plot is flipped. Can't wait to see where this goes in book two.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on July 7, 2023
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