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Description
Rat CASP12 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 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. 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 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 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 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/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 a caspase 12 (CASP12) 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 caspase 12 (CASP12) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Caspase 12 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Caspase 12 (CASP12) is a protein encoded by the CASP12 gene. This protein belongs to a family of enzymes known as caspases, which cleave their substrates at the C-terminal aspartate residue. It is closely related to caspase 1 and other members of the caspase family, known as inflammatory caspases, which process and activate inflammatory cytokines such as interleukin 1 and interleukin 18. This gene is polymorphic, producing either the full-length caspase protein (Csp12L) or an inactive truncated form (Csp12S). | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, and other biological fluids |
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4.3 ★★★★★
Based on 1376 reviews
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Product Reviews
★★★★★ 4
Awesome, but not the best squeakers.
Color: Purple
I bought the orange gecko for my 5-month-old Corgi puppy. He absolutely loves it. He came up with a "game" (sort of) where if I pick it up and start squeaking one of the ends/squeakers really quickly over and over, he'll grab the other end/squeaker and start doing the same thing with me. It was really cute until one of the squeakers stopped working about a week after I bought it... but I guess the toy wasn't too expensive, and he doesn't really seem to mind that one of the squeakers doesn't work anymore. I might buy another next time I do an Amazon order (so I can get the free shipping) and see if it lasts a little longer.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on August 2, 2013
★★★★★ 5
Great Toy For a Great Price
Color: Purple
My Aussie loves toys and plays with them as though she is a small child. She has a basket and will go to the basket and dig through it until she finds what she is looking for. Lately she has been using the bungee gecko as her toy of choice. She gets bored easily and will rip the stuffing out of most toys, the fact that this one has lasted 6 weeks or so is impressive with no rips, holes or missing limbs. Her favorite way to play is to come over to me and nudge me to play tug of war and we have broke many toys this way but so far we can play tug a war several minutes at a time and the gecko is still in one piece. For the price which is considerable less than some of her other toys I have already ordered a couple of replacements for the future. This is a great well built dog toy that has has a lot of give and can be stretched without ripping.
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Reviewed in the United States on May 3, 2013
★★★★★ 3
No longer as durable
Color: Purple
I have one of these that has made it through 2 dogs but now the bungee is stretched out. It's been such a well loved toy I recently got another one. They've changed the plush fabric. The old one was indestructible. This new one is much thinner and easily torn. I wish they would go back to the old plush fabric.
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Reviewed in the United States on February 4, 2024
★★★★★ 5
Great Toy!
Color: Orange
This has been a great toy for our 11 week old German Shepherd. She absolutely loves it. I can get out all of her toys and lay them out with the exception of her "Lizzy" and she will immediately go to her toy bin and get it herself. Needless to say it is her favorite toy. She has had it a week now and she plays with it a lot, chewing and squeaking. It's a pretty amazing toy to have held up this long already and to date shows no sign of wear or damage. Lizzy just keeps going and going.
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Reviewed in the United States on April 4, 2014
★★★★★ 1
This item came with plush missingand thread unraveling on one leg
Color: Blue
I bought this toy because my dog spent a week playing with the same toy from Zanies that belonged to a visiting dog. My dog was very gentle with this toy, which he loved, so we decided to find one for him. The toy that arrived had no Zanies label and missing plush on the bottom where the label should have been, plus other bare spots in the area that showed the white mesh beneath. It also had a long thread at the joint of a rear leg so I knotted it at the joint and clipped it to prevent my dog from pulling at it but it's apparent that the sewing is loose and wont last long. The color was also much lighter than the toy he'd been playing with that belonged to the guest dog but I figured it might just be from a different dye lot (it was not bought at Amazon but their local pet store.) I am not convinced this is a real Zanies toy. There were two labels in French and it said it was made in China. I am returning this toy and will get a replacement and see how it looks. If it seems like it will fall apart or is not the real thing I will return it immediately. If it has a label and is in good shape and sewn well at the seams I'll revise this review.
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Reviewed in the United States on August 4, 2020
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