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Description
Human MLCK 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 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 Myosin Light Chain Kinase (MLCK) capture antibody. 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 Myosin Light Chain Kinase (MLCK) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Myosin Light Chain Kinase ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Myosin light chain kinase, also known as MYLK or MLCK, is a serine/threonine-specific protein kinase that phosphorylates a specific myosin light chain, the regulatory light chain of myosin II. It plays a crucial role in muscle contraction. Smooth muscle fiber contraction can begin upon the influx of calcium ions (Ca2+) into the muscle from the sarcoplasmic reticulum or extracellular space. Initially, calcium binds to calmodulin. Following this influx of calcium ions and its binding to calmodulin, pp60 SRC (a protein kinase) induces a conformational change in MYLK, activating it and leading to increased phosphorylation of myosin light chains at serine residue 19. | |||||||||||||||||||||||||||||||||
| 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, and other biological fluids |
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4.4 ★★★★★
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Product Reviews
★★★★★ 3
You get what you pay for
Style: Steam Mop
It's just OK. It does get the job done, but poorly made and I don't expect it to last long. Unlike other steam mops I've had, this one leaves the floor wet. Plus the pad does not pick up any stuck-on gunk that the steam loosened. I had to go back and pick up a few pieces by hand. Instructions say to let the water tank air dry before storing, but the attached lid makes it hard to let air into the tank. In retrospect I should have spent a few dollars more and gotten a better unit.
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Reviewed in the United States on April 22, 2026
★★★★★ 5
Good purchase for the price point
Style: Steam Mop
This mop is perfect as it is compact while being easy to use. Stuck food on the floor? Not after using this. Crayon? Vanishes. Sticky substance? no more. Great but for the price
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Reviewed in the United States on May 31, 2026
★★★★★ 5
It is a keeper for sure!
Style: Steam Mop
This is about 100% better than I ever imagined. It has every feature I want and none of the stuff I don't want. It has a SUPER long cord, it heats up quick and works exactly as it is supposed to. I steam cleaned everything from my stained concrete floors, THE ENTIRE COUCH, and low pile area rugs. Love this thing and SO MUCH cheaper that the competing brands.
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Reviewed in the United States on January 31, 2026
★★★★★ 5
Works!
Style: Steam Mop
It’s so nice and works as expected. My mother in law switched hers for this one.
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Reviewed in the United States on June 3, 2026
★★★★★ 5
Great Starter Knife Set With Everything You Need
Size: 9-Piece, Style: Traditional Handle Design
5/5 stars for the Amazon Basics High Carbon Stainless Steel Knife Set. I have been really impressed with this set overall and it has handled pretty much every kitchen task I have thrown at it so far. Right out of the box the knives were insanely sharp and felt much higher quality than I was expecting for the price.
One of the biggest pros is the variety of knives included. It comes with basically every knife you would need for normal cooking and kitchen use which makes it a great all-in-one set. The knives also have a really nice weight to them and feel balanced in the hand instead of feeling cheap or flimsy. The grip is nice as well not slippery and while using it I feel like it stays put in my hand. They have held up well with regular use and have been easy to clean after cooking. The high carbon stainless steel has also done a good job resisting rust and staining which is important for a set that gets used often.
Another nice addition is the included honing rod which helps maintain the edge and keep the blades performing well over time. I did notice that the edge on the knife I use the most has started to wear down a tiny bit after extended use, but that is expected with pretty much any frequently used kitchen knife and can usually be maintained with regular honing.
As for cons, the wooden knife block is nice looking but I could see it potentially cracking over time if it is not taken care of properly or exposed to too much moisture. Other than that, I really do not have many complaints with this set.
Overall I think this is a fantastic knife set for anyone who just moved out and needs a complete kitchen setup or for someone wanting to replace an older worn out knife set. I actually gifted a set to my in-laws already and they have been loving it as well. I would definitely recommend it.
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Reviewed in the United States on May 28, 2026