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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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4.7 ★★★★★
Based on 22 reviews
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Product Reviews
★★★★★ 5
Great find for my dog!
Color: Hedgehog, Style: Big Squeak Hedgehog
This is a Big squeaking toy And has become a favorite of my dog. He is a big chewer but he won’t chew at this one he just carries it around and plays catch with it. I believe the little spikes keeps him from heavy chewing on it! Great find for us! But it is a loud squeak!
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Reviewed in the United States on March 26, 2026
★★★★★ 5
For the love of a dog!
Color: Blue, Style: Tootiez Hedgehog, Color: Blue, Style: Tootiez Hedgehog
Okay, first - this toy is a hoot. When you first get it and hear it's cute grunting/tooting sound, you can't help but grin and think, Yup! That sounds like somebody in here just tooted! Which for some reason always does seem to be a bit funny, doesn't it? And you'll probably find yourself chuckling a little and saying, Oh how cute. After that, the next logical step is you'll introduce the toy to your dog. And then, depending on your dog's particular personality, you may soon discover (as we did) the amazing love/hate relationship a human can develop with a simple dog toy.
We have a 1-yr-old standard poodle whom we named Kenda. And yes, he is named after Joe (for any of you ID fans out there). His official AKC registered name is Lieutenant Kenda, Home Inside Hunter. Corny? No doubt. But it truly seemed an appropriate name for him, because this is the first dog we've ever owned that actually LOVES to play with dog toys and will endlessly hunt them down throughout the house. No toy, however well hidden, stands a chance with this determined toy hunter. As Joe might say, he WILL find you! :) His toys are his friends, and he is fiercely devoted to them.
Enter the adorable little rubber hedgehog with his even more adorable "toot". The moment Kenda laid eyes (or ears?) on this little guy, all other toys were forgotten. It was love at first sight. So much so that within a few hours of him playing with this toy to the exclusion of all others, we decided to give him a name. We call him "Blue" (I know, we're so creative). Blue immediately became Kenda's best friend - or at least his best toy. He played with him constantly. He bit him, he wrestled with him, he chewed on him. He brought Blue to us and, if we were sitting down, very carefully placed this slobbery ball of rubber in our laps, as if asking, Can we play catch with Blue? Huh? Pretty please?? Sometimes we did, sometimes we didn't. On those occasions when we didn't, he would play catch with himself, picking Blue up in his mouth, swinging his head, and tossing him across the kitchen; then running/sliding across the kitchen floor to retrieve him on the other side of the room. In the beginning, if Blue was nowhere in sight (and with dogs, out of sight is usually out of mind), the hubby and I would get a kick out of saying, "Kenda, where's Blue?!" Just for the enormous fun of watching a 55-pound poodle suddenly leap a foot in the air, scramble his legs mid-air like Fred Flintstone getting his car started, then half running/half sliding across the kitchen hardwood floor in a desperate effort to find his beloved Blue. Oh, how we entertained ourselves in those early days watching Kenda with his Blue. And through it all, through every bite, squeeze, toss, push, throw, and chew of this toy.......the toot. The grunt. Okay, let's call it what it really sounds like, folks: a FART, okay? There, I've said it. It sounds like your grandpa just passed gas - bigtime. Funny? At first, yes. Hilarious. But a thousand times a day? Over and over and over? While you're trying to talk on the phone? While you're trying to have conversation with each other over coffee at the end of the day? Sometimes for an hour NON-STOP? Well, let's just say the humor of it all began to elude us a bit. And therein lies our love/hate relationship with this adorable little toy.
We thought we'd died and gone to heaven one day when Blue stopped tooting. Turns out Kenda had chewed on him so much his tooter (located rather anatomically correctly in his tushie) had fallen out. Or rather IN, since it was now in Blue's tummy. Poor Blue, he couldn't make noise anymore, and although Kenda kept playing with him you could tell he was confused as to why his little buddy had fallen silent and wouldn't "talk" to him anymore. And as much as the hubby and I were enjoying the tooting reprieve, we couldn't take it. By the third silent day, I could almost feel the invisible hands of Amazon coaxing me toward my computer, gently urging me to buy another Blue. But I resisted, folks. I did NOT buy another Blue. I bought TWO more Blues! One for now, and one for that possible future day when this Blue, too, falls silent. Why? Because ... well, because it's BLUE! He's practically a member of the family now. The dog loves Blue, and we love the dog. I guess it's that simple.
My final word on this dog toy? It's adorable. It's well made and will hold up to a ton of play and chewing. His tooter may not survive as long; I guess that remains to be seen. And if your dog is anything like mine, well then your sanity may take a hit as well. But if your dog loves his little hedgehog buddy as ours does, and if you love your dog (and you know you do!), then you might decide your sanity is worth the risk.
Two thumbs way, WAY up!
P.S. Blue now has a friend. We just bought the pink sheep. Kenda is in 7th heaven. Our house sounds like a retirement home after a chili bean supper. And yes, we named him "Pink". I told you - we are nothing if not creative.
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Reviewed in the United States on June 6, 2018
★★★★★ 5
Strong hold
Color: Multi-colored, Color: Multi-colored
Very good product. I’m definitely impressed. I have two German shepherds and this toy is definitely durable and does what it’s made to do. The suction is great. The company even gives you a clear plastic strip that you can actually put on the floor and then put the suction cup on it For better grip. I’ve had suction toys before which was absolutely horrible. This one definitely supersedes all others. The suction comes off once in a while but usually that’s because if my dogs are literally biting on the suction cup part lol. But with confidence. Great product
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Reviewed in the United States on February 23, 2024
★★★★★ 5
Absolutely recommend for active dogs or puppies
Color: Multi-colored
Absolutely recommend! I have a 10 month old puppy and he has been obsessed with this since the day I bought it a few months ago.
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Reviewed in the United States on May 17, 2026
★★★★★ 4
Base of you is very durable
Color: Blue
Overall this is a fun toy. My German Shepherd loves it. The ropes were chewed off within the first hour which was disappointing. Using it for treats did not work because the opening is so large. It has become only a chew toy and it is very durable. Since I bought this to enjoy the extra features, I am disappointed. I was able to buy three chew only toys for the same price. I still give it four stars because my dog likes it so much.
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Reviewed in the United States on March 15, 2023