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Description
Mouse PKA 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 the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. Cell lysis solution: Gently wash adherent cells with pre-cooled PBS, then digest with trypsin, and collect the cells after centrifugation at 1000×g for 5 minutes; suspended cells can be directly collected by centrifugation. Wash the collected cells 3 times with pre-cooled PBS, add 150-200uL PBS for every 1×10^6 cells to resuspend (it is recommended to add protease inhibitors to PBS; if the content is very low, the PBS volume can be appropriately reduced) and break the cells by repeated freezing and thawing or ultrasound. Centrifuge the extract at 2-8℃, 1500×g for 10 minutes, and take the supernatant for detection. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, take the supernatant for detection, or store the supernatant at -20℃ or -80℃, 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 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 protein kinase A (PKA) capture antibody. After incubation and washing, the assay 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 protein kinase A (PKA) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Protein kinase A | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Protein kinase A (PKA) is a family of enzymes whose activity depends on cyclic AMP (cAMP) levels in cells. It is also known as cAMP-dependent protein kinase (EC 2.7.11.11). PKA has multiple functions in cells, including regulating glycogen, sugar, and lipid metabolism. It is one of the most extensively studied protein kinases, in part due to its uniqueness. Of the 540 distinct protein kinase genes that make up the kinome, only one other protein kinase, casein kinase 2, exists as a physiological quaternary complex, consisting of four subunits. | |||||||||||||||||||||||||||||||||
| 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, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids |
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4.0 ★★★★★
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Product Reviews
★★★★★ 5
User friendly and I highly recommend
Color: White
I recently purchased the Manvins Robot Vacuum and Mop Combo with WiFi capabilities, and it has been a game-changer for keeping my home clean. The setup was incredibly easy, and I was able to connect it to my smartphone in minutes using the app. Once connected, I can schedule cleaning sessions, monitor progress, and control the vacuum and mop remotely, which is super convenient.
The vacuum picks up dust, pet hair, and debris effortlessly, even from carpets. The mop function is a nice bonus, and it does a great job of leaving floors streak-free. I was surprised at how well it handled both tasks simultaneously, and it’s great to not have to worry about switching between modes
The WiFi feature really shines when I’m away from home. I can activate the cleaning process from anywhere, and it returns to its charging station when it’s done. It even maps out my home as it cleans, ensuring that it doesn’t miss any spots.
Overall, this robot vacuum and mop combo has made cleaning much more efficient, and I love how easy it is to keep my floors spotless with minimal effort. Highly recommend it for anyone looking for a smart, reliable cleaning solution!
Sent from my iPhone
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Reviewed in the United States on December 30, 2024
★★★★★ 5
Excellent vacuum!!
Color: White
This is a hard working little robot. It cleans dirt and debris so easily and even gets the Arizona dust that accumulates everyday. I have a cat the sheds and 4 boys plus my husband and this vacuum works like a champ. I haven’t tried the mop feature yet because I just haven’t sat down and read the directions. I’ll update when I try that feature out. I love that it can go under my furniture and climb up rugs, even my cat scratch boards. I 100% recommend this vacuum. It has changed my house keeping routine and saves me a ton of time. I absolutely love it and you can’t beat the price!
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Reviewed in the United States on November 15, 2025
★★★★★ 5
Pleasantly surprised.
Color: White, Color: White
Based on the price I wasn't expecting much but boy was I wrong. I'm so impressed and thrilled with this purchase. I have a long haired Maine Coon and it picked up so much fur. It picked up thread, small pieces of yarn and feathers from my pillows. I watched the long front brushes pull dust and dirt out from under spaces that my regular vacuum would never get without moving things. I have carpet, hardwood, throw rugs and even a long shag throw by my bed that it maneuvered over mostly easily. I helped it over one or two transitions but it never got stuck. Just turned and kept going. The mop worked great. Only concern is that it only uses water so it's not a deep clean but the pad was dirty and once it dried I threw it into the wash. Definitely will use it in between a deeper cleans making cleaning a whole lot easier. With a cat shredding the carpet on a scratching post and all of his fur I was shocked how packed the dirt cup was and how great the house looked with all the little bits of scratching pole, oh yeah and card board scratching pad cleaned up so easily. I highly recommend. The cat thinks it's a cat toy so he kept starting it up. I had to cover it to protect it from the cat. It now lives under it's very own plastic bin. 🙂
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Reviewed in the United States on March 13, 2025
★★★★★ 5
Robotic Vacuum
Color: Jet Black, Color: Jet Black
Good Cleaner
I'm very old and it was very difficult for me to clean my home but thank God my Daughter told me about Robotic Vacuum cleaners so, I ordered it and now I'm using it, really impressive results.
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Reviewed in the United States on April 22, 2026
★★★★★ 5
My first experience with a Robot vacuum
Color: White
I am liking this robot vacuum. It is very easy to program and can be done from your phone. It has mapping capabilities which helps with learning the room. The remote is easy to use too. It picks up most dust particles and pet fur from rugs and bare floors. It has a wet floor mopping pad which is so helpful. Surprisingly it picks up more than you think. The dust cup holds quite a bit before you have to empty it. The little side brushes help with collecting the particles. It can go under some furniture and along the walls. You have several floor patterns that it can be programmed for. It is very quiet too. Recharging doesn’t take long either. It can even be programmed to vacuum while you are away. Instructions are downloaded through the app.
My cats are very curious about how it moves about the rooms. So much fun to watch them.
I really got a great bargain for this robot vacuum on Amazon.
(I found that if you have clutter on the floors or cords under furniture you should remove and secure them before vacuuming.) It will find a way to go from floor to area rugs once it maps out the area.
I am still learning the functions in the app👍🏾👍🏾😃
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Reviewed in the United States on January 9, 2025