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Description
Rat pf1 ELISA KitProduct Specification Usage Required experimental equipment: 1. Microplate reader (450nm) 2. High precision pipettes and pipette tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37C incubator 4. Distilled or deionized water Sample preparation and requirements: 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 and
Product Specification
| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample preparation and requirements: 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 and mince the tissue. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL 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 collect the supernatant for analysis. Cell Lysis Buffer: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled 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 freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freezing and thawing. 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 perforin 1 (pf1) capture antibody. 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 perforin 1 (pf1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Perforin 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Perforin-1 is a protein encoded by the PRF1 gene. Perforin is a pore-forming cytolytic protein found in the granules of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). During degranulation, perforin molecules are transferred to target cells with the help of calmodulin, a chaperone protein that protects perforin from degradation. Perforin then binds to the target cell's plasma membrane via membrane phospholipids. Phosphatidylcholine binds to calcium ions, increasing perforin's affinity for the membrane. The resulting pores allow a family of pro-apoptotic proteases, known as granzymes, to passively diffuse into the target cell. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.6 ★★★★★
Based on 126 reviews
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Product Reviews
★★★★★ 1
Not the same rubber.
I just received these today. I already have two of the same size direct from the local pet store. The one thing I liked about them that made me want to buy more - my 13lb dog could chew on them all night and never leave a mark. The red one I received was as expected. The blue one I received was not made out of the same rubber. It was softer. We played catch a few times and it didn’t bounce the same. I looked away for 5 minutes and she had it already starting to shred. Not sure what happened but 1 of the 2 balls I was sent was not up to par, possibly defective.
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Reviewed in the United States on May 23, 2024
★★★★★ 5
Super Sturdy with hours of fun.
Color: Wild Duck, Hare, Squirrel & Alligator, Size: Small
I got the small ones that you cannot put a water bottle in.
These toys have held up way better than expected. I am dog sitting a very rough with toys dog named Chunk. He tears up ever. Single. Toy my dog (Ted) has.
I bought new ones just for chunk. These appealed to me with the no stuffing, They were cheaper, and have a squeaker. Everything chunk needed. They arrived on time and I thought I wasted my money. They were super soft, like too soft. They felt flimsy and weak. Like 5 minutes with chunk and they be dead. 2 hours with my dog and they would have a rip. So far 2 of them (I set two out) have lasted the whole weekend. The bunny looks very rough and disgusting, but no rips, the squeakers still work, and no random fluff everywhere. I am going to wash the bunny once chunk leaves.
My very careful doggo Ted loves them too. He throws them up in the air and catches them, plays tug of war with them, and no rips. They are super sturdy. He can’t wait until Chunk leaves (chunk is 2 and Ted is 12-13) so he can play with them.
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Reviewed in the United States on April 26, 2026
★★★★★ 5
Loved it
Color: Brown/Wild Duck, Size: Large, Color: Brown/Wild Duck, Size: Large
He loved it so much, he destroyed it so we ordered another.
I didn't realize that you can stick an empty water bottle inside.
Again, the first one didn't last long, but he enjoyed it
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Reviewed in the United States on May 20, 2026
★★★★★ 5
Very fun interactive toy
Color: Brown/Wild Duck, Size: Medium
This toy is very versatile. There's no stuffing yet it's very soft so it can be used as a tug toy. It also makes for a fun fetch toy because it has a cavity to put a water bottle. It gives your dog something that crinkles and can be pierced when bitten and easily replaced with another empty bottle whenever necessary.
That is what really makes this toy the standout of all the ones offered by Best Pet imo. I don't expect this to last forever but once my pup gets bigger we'll probably get the large size for her because there's not too many all in one fetch and tug toys at an affordable price.
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Reviewed in the United States on May 15, 2026
★★★★★ 4
Great Toy, but Big Dogs Destroy the Squeakers Fast
Color: Wild Duck, Hare, Squirrel & Alligator, Size: Large
My big dogs had the squeaker torn out in no time, but my little dog absolutely loves this toy. It’s the perfect size for her, and she carries it everywhere. Durable enough for small pups, just not tough enough for heavy chewers. Still a fun toy overall!
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Reviewed in the United States on February 15, 2026
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