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Description
Mouse MLKL 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 harvest cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Wash the harvested 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. Pre-Assay Preparation: 1. Remove the kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/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 capture antibody against Mixed Lineage Kinase Domain-Like Protein (MLKL). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of Mixed Lineage Kinase Domain-Like Protein (MLKL) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Mixed Lineage Kinase Domain Like Protein ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Mixed lineage kinase domain-like protein (MLKL) is a protein encoded by the MLKL gene. This gene belongs to the protein kinase superfamily. The encoded protein contains a protein kinase-like domain. This protein plays a key role in tumor necrosis factor (TNF)-induced necroptosis, a programmed cell death process, and inhibits TNF-induced necroptosis through interaction with receptor-interacting protein 3 (RIP3). | |||||||||||||||||||||||||||||||||
| 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 | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenate, cell lysate |
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4.4 ★★★★★
Based on 6 reviews
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Product Reviews
★★★★★ 5
My Dogs Favorite for throwing
Color: D) 2-Pack Lattice (3.25" Balls), Color: D) 2-Pack Lattice (3.25" Balls)
We brought our new adoptee home end of July, and I quickly had to find him replacements for his tennis balls because those balls are inherently abrasive and our yard has sand which gets in the fuzz and makes them extra abrasive and they were already causing tooth wear (our previous shepherd chewed tennis balls like gum for a week after his adoption, wore his teeth tips enough I noticed and I took them from him too but this guy didn't like the hand me down replacement, it was too big for his preference so I had to hunt down different ones for the current dog).
I tried the glow in the dark Chuk It ones, but he doesn't like to carry them, or the smaller regular chukits... My current dog prefers these web style ones. I like glow in the dark stuff and it makes them easier to find in the evening or over night (if you have night adjusted eyes and not a lot of lights in the yard these are VERY glowy at night). Now I know he likes them I am thinking winter is coming and I may lose the glow color in the Michigan snow, so I'm here to order some in some in non snow camouflage because I want to be able to play in the winter without as much groping for hiding balls (literally he will chase and then snub any other balls in the yard I throw except the huge jolly ball but that isn't a fetch type ball, its a thrash toy. these are the only ones he will actually return for me to swap and throw indefinitely).
I included video and a picture of them at the local beach. They float with enough above the water for maybe a few ants to use them as a flotation device, but take a bit of time to come to the surface when they splash down. I think brighter colors will help make them easier to find in the water as well as the snow.
I think my dog likes them because the webbing design makes it easier to grip, even when slobbery his teeth can get a hold in the gaps (so can fingers haha) without slipping too badly. They also compress almost flat and boing back into shape and seem to be durable over at least 6 weeks of summer living in the yard, so far so good. They don't have a huge amount of mass so they don't whip super far but if you get a technique down you can get very reasonable distance on throws. He doesn't gnaw on them, so I can't speak to resistance to actual shearing attempts with teeth, just grab and chomp for fun, then holding in his mouth.
My dog weighs about 68 lbs (we are trying to gain a bit), he is a German Shepherd, and this size fits him well (can't remember which size I got but usually Amazon shows what size the reviewer got near the title of the review. They're big enough I'm not worried he will accidentally swallow or choke on them, small enough his mouth fits them comfortably, his grip is secure because of the gaps, and he loves them. I suggest get two at least unless your pooch is super good about dropping it and letting you pick it up. With two, I can whip a ball, find the one he just dropped, and by the time he is back, I have the other one in hand. He knows I won't throw it unless he drops the one he just returned (I will wait for him to remember this), and this makes it way easier to play thow and bring it almost back (he often drops it at speed when he is nearly back to me so it rolls past me) without playing chase me for the toy... and makes reinforcing Drop It as an safe thing to do, like he isn't going to lose out if he drops the fun ball in his mouth, I have the second ball to mark (say YES!!! when he drops it) and immediately reward by throwing the ball I already had... he loves them enough that he thinks other balls are stupid so I don't want to throw a less cool ball to exchange for the cool ones haha
Anyway hope this was helpful, Im off to order some of these balls in brighter colors for lake and snow visibility.
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Reviewed in the United States on September 20, 2024
★★★★★ 4
Fun Glow Balls for Night Play, Great Value
Color: A) 2-Pack (2.5" Balls)
The Chew King 2 Piece Glowing Fetch Balls are a solid, budget-friendly alternative to Chuck-It glow balls. While my pups prefer the mouth feel of the Chuck-It balls, these still provide bright, visible fun for dusk or nighttime play, and can even be used indoors with a flashlight to get the game going.
Durable, glowing, and engaging—a good option for evening fetch sessions which always insures that our pups get a great night of rest.
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Reviewed in the United States on January 6, 2026
★★★★★ 5
Durable, Perfect Size, and Glows
Color: A) 2-Pack (2.5" Balls)
I bought these for my 23 pound Bojack (Boston Terrier/Jack Russel) mix and he absolutely loves them. He’s an aggressive chewer that has destroyed Kong toys. This ball has just enough flex or give in it that he can’t tear it apart as it compresses in on itself. After about 6 months it started to wear down and tear a bit by the hole so I just tossed it and gave him the other one. I’ll be buying more of these as they wear out over time.
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Reviewed in the United States on May 14, 2026
★★★★★ 5
A Must for Nighttime Fetch!!
Color: A) 2-Pack (2.5" Balls)
These are an absolute Must!! We have an ESS who is nearly 2yo and destroys Every toy he has. For some reason, he doesn't try to annihilate these glow balls. If he chomps on them, it's just for a moment while he is bringing it back to you. Has just a little weight to them so it goes far enough when thrown and a nice bounce when we have to play in the house, due to weather. Wish the bright 'glow' held a little longer but stays dim for quite some time and overall, just Awesome! Also purchased the 'chuck it' brand and sent them back because these were so much better. Will be buying more, if ever needed, but haven't had to because they are So Durable.
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Reviewed in the United States on April 12, 2026
★★★★★ 5
Awesome quality glow balls
Color: I) 8-Pack (2.5" Balls)
I have bought every glow ball on amazon many times over for my german shepherds. My longest lasting balls have been the chuck-it, they seem to glow longer and not get destroyed but are expensive. These glow balls are our new favorite and not only because of the low price. They hold a good glow longer are heavy duty and fit the chuck-it thrower perfectly. I am stocking up on these. Shipping was also next day so it's 100% buy on these.
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Reviewed in the United States on February 8, 2026