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Description
Rat CAT 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. 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 50 ng/mL). Then dilute to the following concentrations: 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL, 1.5625 ng/mL, 0.78125 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 50ng/mL standard working solution into the first EP tube and mix thoroughly to make a 25ng/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 a Catalase (CAT) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of Catalase (CAT) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Catalase ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Catalase is a common enzyme found in almost all organisms exposed to oxygen, such as bacteria, plants, and animals, that catalyzes the decomposition of hydrogen peroxide into water and oxygen. It is a very important enzyme that protects cells from oxidative damage caused by reactive oxygen species (ROS). Catalase has one of the highest turnover rates of all enzymes; a single catalase molecule can convert millions of hydrogen peroxide molecules per second into water and oxygen. Catalase is a tetramer composed of four polypeptide chains, each with over 500 amino acids. It contains four iron-containing heme groups that enable the enzyme to react with hydrogen peroxide. | |||||||||||||||||||||||||||||||||
| 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 | 0.78-50 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates and other biological fluids |
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4.5 ★★★★★
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Product Reviews
★★★★★ 5
No comment
Format: Hardcover
My grandkids loved it I read it over and over to them
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Reviewed in the United States on April 20, 2021
★★★★★ 5
Grandson loves this book & so do I!
Format: Hardcover
Great book! My 5yr old grandson cracks up when we read it.
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Reviewed in the United States on October 20, 2020
★★★★★ 5
Wonderful!
Format: Hardcover
Absolutely hilarious for all ages
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Reviewed in the United States on September 12, 2021
★★★★★ 5
Father and son both have a great imagination
Format: Hardcover, Format: Hardcover
I think Little Fox showed his father, Poppa Fox that he also some great imagination in this cute picture book by Jorma Taccone. I had to laugh as I read this book as I could totally relate to how Poppa Fox felt. As a parent, or just a human being, you feel like you’re doing the right thing, having fun and then bam, you’re thinking aww, what just happened?
I thought it was sweet when Poppa Fox played pretend with Little Fox on their way home from school. Having a rough day at school, Little Fox needed something to make him happy again and his father was doing just that. Using father’s imagination, the two of them, pretended to be race cars as they raced off to the bus stop. As the night wore on, this game of pretend continued, only now Little Fox was getting more involved. What started out as a fun, friendly game was now turning violent, messy and involved, leaving Poppa Fox picking up the pieces.
I liked how the book progressed from a fun friendly game to an intense involved event. I think the illustrations fit perfectly with the text as you could see this transformation and the drama unfolded right before your eyes. The text fonts were great too as they varied in sizes, shapes and colors depending on the situation. I enjoyed Poppa Fox patience as he handled the situation with his son, Little Fox. I had to laugh when Little Fox wanted his father to read to him 5,000 books before he went to bed. Now, wouldn’t that be a nice library for a child to have in their house? There is some violence in the book as they play pretend, so you may want to consider this, if that bothers you but it is pretend. I thought this was a fun, entertaining read and I highly recommend it.
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Reviewed in the United States on December 2, 2020
★★★★★ 5
Really magical!
Format: Kindle
I loved everything about this book from the illustrations to the all the scenarios. I especially loved Poppa Fox's and Little Fox's relationship. A wonderful father-son duo. 😊
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Reviewed in the United States on September 27, 2020