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Description
Human 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: Adherent cells should be gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells should be washed three times with pre-chilled PBS and resuspended 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. 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 enzyme conjugate are sequentially added to microwells pre-coated with MLKL capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of MLKL in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human MLKL 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 necrosis 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.4 ★★★★★
Based on 24 reviews
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Product Reviews
★★★★★ 3
Great idea but didn't hold up!
Size: Large
My dog and I really enjoyed this as a tug toy to use where it kept my hands away from accidental nips. When we initially got it we let her play solo with it after our tug session and I noticed that she left little teeth puncture holes so we packed it away for awhile and exclusively used the other new toy we bought for her. After a few weeks we took this back out and, again, we loved it and this time I allowed her to play with it longer and although it was showing a lot of the little tooth marks it seemed to be holding up so I left it out with her. None seemed deep, just seemed like impressions from her teeth.
We continued to enjoy it for about a week where she'd carry it over to us for tug sessions, loop it on our feet when we were sitting watching tv and chewing on it a bit. I'd look at it when we'd tug and, like I said above, it seemed to be holding up. After one session of tug when she took it off to chew on and I was watching some tv I checked on her after awhile and she had bitten through the black side and had chewed up into small pieces about a two inch section of it. Small, little hard bits were in a pile on the floor. She didn't just bite through, it had broken into small, little pieces and hopefully I was able to get all of them and she didn't swallow any!
So if you want to get this exclusively as just a tug toy and not something for your dog to chew on and plan on putting it away after I think it will be okay for that and should last but if your dog is a serious chewer like ours is you may not want them to be allowed to chew on this. Seemed like it was going to last and then suddenly I was throwing away half of it and hoping she didn't eat any of the black bits. We still have the yellow side and will take that out for some tugging but no longer for her to play with freely!! She did tremendously love running around with this thing and would flip up the loose end and hook it on her face or our feet and was so cute to watch her!! Wish it had lasted!!
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Reviewed in the United States on May 9, 2013
★★★★★ 5
Name holds true.
Size: Large Triple
Just bought my second set of these.. first set lasted my healer almost 4 years and are still together but starting to split at the smaller seams so you can’t play as aggressive as my dog would like. We bought the second set and now she has her second wind! She loves tugging and I love knowing this toy will last a good while especially with an active dog!
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Reviewed in the United States on January 4, 2025
★★★★★ 5
The dogs like it very much, it produces a lot of noise.
Size: Large, Size: Large
I’m rating this based on my dogs’ response to this toy – they absolutely love it, and it has seen more use without damage than any of their regular throwing toys. At the same time, unless you enjoy, REALLY ENJOY the sound of crunching water bottles, you may not like having this ball in the house.
Pros:
The rubber outer shell of the ball is durable, and can survive both chewing and pulling forces (as a note, my dogs do not concentrate on chewing - they use it more for play games). It’s big enough for my dogs to play tug of war with it, hooking their teeth in the mesh, and they since it bounces around if they drop or kick it, it makes the game more interesting than rope. They did crush the interior plastic, but they don't really seem to care.
It’s much more engaging than a normal ball, and can be modified to hold treats, sticks, rope elements and other external bits, which my shepherd mix loves; he gets bored easily and frequently, and this can actually entertain him.
Cons:
This toy is loud, and produces repetitive crunching sounds. It is apparently really engaging to chew, and my dogs will produce the horrid plastic crackling up to half an hour at a time. If you let your dogs in and out, it would make a fine outdoor toy, but since I have a dog door, it migrates around to pretty much wherever the dogs are. I kind of hate it, but will probably buy another if they break this one since they enjoy it so much.
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Reviewed in the United States on October 17, 2016
★★★★★ 5
Very durable and my Heeler's fave inside toy
Size: Large
My red Heeler loves these balls, second only to Tennis balls but she destroys those within minutes and they are outside only. This is her inside ball. She is hard on toys and has the strongest bite of any dog I've had (shepherds, collies, weimaraners, retrievers, Labs) yet these continue to last. She catches the ball, we play tug of war with it (she places it in hand hand and pulls only when I have a firm grip, she shakes it and flings it. The rubber outside even outlasts the water-bottle like plastic piece inside. Steps holds the ball with her foot and pulls on the rubber with her mouth, stretching it to sometimes twice its size. I pull while shes pulling. This thing gets abused daily and keeps on going. Its the most used toy and she brings it to me to play with every day. Each ball lasts about 6 months before the inside ball is toast and the outer rubber is finally starting to tear/break. which for me in an incredibly long time. If this ball is available to play, she wants it over all others. Shes NEVER gotten a piece off to actually swallow and that alone is worth the price.
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Reviewed in the United States on June 4, 2025
★★★★★ 4
Good for price
Size: Medium
Good product, wish it was a little more chew resistant
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Reviewed in the United States on April 29, 2026
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