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Description
Rat TWEAK 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 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. 3. 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. 4. 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 the collected cells three times with ice-cold PBS and resuspend them 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 collect the supernatant for analysis. 5. 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. Preparation of 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 2000pg/mL). Then dilute to the following concentrations: 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 2000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 1000pg/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 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 a capture antibody against tumor necrosis factor-related weak apoptosis-inducing factor (TWEAK). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of tumor necrosis factor-related weak apoptosis-inducing factor (TWEAK) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Tumor necrosis factor-related weak apoptosis-inducing factor ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Tumor necrosis factor-related weak inducer of apoptosis (TWEAK), also known as tumor necrosis factor ligand superfamily member 12, is a protein encoded by the TNFSF12 gene. The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This protein is a ligand for the FN14/TWEAKR receptor. This cytokine has overlapping signaling functions with TNF but exhibits a more widespread tissue distribution. | |||||||||||||||||||||||||||||||||
| 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 | 31.2-2000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell lysates and other biological fluids |
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4.8 ★★★★★
Based on 5 reviews
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Product Reviews
★★★★★ 5
Fun Story For Any Age Fan
Format: Paperback
Bruce Wayne is the only kid at his middle school without some kind of special powers. He wants to make a difference in Gotham but he thinks he doesn’t stand a chance compared to all of the other students with impressive skills. Bruce Wayne: Not Super follows Bruce’s journey to finding out what makes him special.
This middle grade graphic novel is very well done. I was already familiar with much of the author, Stuart Gibbs’ work. His style and sense of humor carry over well to this type of comic. The illustrations pair nicely and are done in a lighter style than more adult Batman comics.
Bruce Wayne: Not Super would be a good introductory book for younger Batman fans or a fun addition for collectors of any age to add to their stash.
I don’t know if the author has worked with DC before but I’d love to see more collaborations in the future for more middle grade graphic novels.
I voluntarily read and reviewed an advanced copy of this book. All thoughts and opinions are my own. Thank you to NetGalley and DC Entertainment (DC Comics)!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 17, 2023
★★★★★ 5
Fun Middle School Origin Story
Format: Paperback, Format: Paperback
Bruce Wayne stands out at his middle school, Gotham Preparatory School for the Really, Really Gifted. No, not because of the wealth he’s inherited from him parents but because he’s the only one without any powers. But when he sees a student bullying another kid, he decides he has to do something. Will he come up with a plan?
This is a fun alternative take on Batman’s origins including cameos from other DC super heroes. The story was entertaining, and I laughed multiple times as I was reading. Be sure to look at the illustrations since some of the jokes are in there. This graphic novel is a very fun read.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 18, 2023
★★★★★ 5
Stunning graphics and a relatable hero
Format: Paperback
As Stuart Gibbs is one of my favorite middle grade authors, I was eager to pick up this book. It did not disappoint. Stunning graphics and a relatable protagonist make this an engaging read. Full of references to the world of superheroes, (some sneaky, some overt) there were plenty of opportunities to make inferences and share a snide chuckle with the author. This title is now on the short list for next year’s Battle of the Books in our school district. Kudos to Stuart Gibbs for the message of brain over brawn (and superpowers)!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 24, 2024
★★★★★ 4
Bruce Wayne: Not Super | Comic Review
Format: Paperback
I recently had the opportunity to check out Bruce Wayne: Not Super from DC Comics. The story is from Stuart Gibbs and the artwork is from Berat Pekmezci, and it is obviously about Batman.
The middle-grade graphic novel follows a teenage Cape Crusader going to school with every other DC character. Heroes and villains all going to school together is a funny concept that I think is pulled off well here.
What’s the joke about Batman? He’s just a rich kid with no powers, so what happens when he goes to a prep school with Superman, Wonder Woman, Flash, Green Arrow, etc.? Bruce has to come to grip with having no powers and how that affects his daily interactions with his classmates.
Bruce’s alienation at being powerless directly conflicts with his goal of being a vigilante hero for Gotham City. His camaraderie with Dick Grayson (Robin) helps him work through his feelings on wanting to be Ferretman Batman.
Aging adult characters down to young teens can come with their own difficulties, but I think Pekmezci nailed it. The artwork is very good throughout this book.
It may not be everybody’s cup of tea, but I really dig these “Elseworlds” stories that place the heroes in completely weird situations. And, what’s weirder than a middle/high school full of super-powered kids!
Clearly, I wasn’t the intended audience for this book, but I think those kids will have a great time reading this.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 6, 2024
★★★★★ 5
Fast read but well loved
Format: Paperback
I gave book 1 and 2 to my 9 year old and she read them both in 2 days and informed me when book 3 will be released. She said she liked the story, the characters had interesting names, and the graphics were exceptional.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on July 24, 2025
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