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Description
Human PKA 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 collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected 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. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, 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 | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Protein kinase A ELISA Kit | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.9 ★★★★★
Based on 26 reviews
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Product Reviews
★★★★★ 3
Why can't they just TALK to each other?!
Format: Kindle
The prologue to this book was bloody and violent. I actually started this book then put it away for several months as I found it not enjoyable. When I finally returned to the book, I pretty much skipped the prologue and went right in. There are a lot of holes in this story, a lot of repetitive sections, and several parts (mostly the whole book) where you just want the lead characters to talk to each other instead of being petty and jealous. Who the female lead is, is never really explained. The humans are considered weak, but the human kingdom is the one they fear, just really weird. I did finish the book and it was overall a good book, and I may actually read the second book to see if any questions are answered and holes in the story filled in. Don't take that as an endorsement, if it was a really good book I would be reluctant to read more as I wouldn't want the story ruined, but just being so-so, there is nothing to ruin and maybe the next will be better. If you're on the fence stay there until you run out of better options, then give it a go.
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Reviewed in the United States on March 25, 2025
★★★★★ 5
A Stellar Dark Romantasy Debut
Format: Kindle
4.5 ⭐️
Yet another stellar debut from a promising new indie author! This dark fantasy romance was a fast-paced, thrilling ride, brimming with high stakes action and slow burn romance. The story was refreshing and original, and I can’t wait to see what this author comes up with next.
There was just enough world building to keep the story interesting, without overtaking the entire story. And I was fully invested in these characters, both the major players and the supporting cast. I loved the sprinkling of humor and playful banter between the whole ensemble. Even though the book encompasses some darker themes, these moments of levity created a beautiful balance within the story.
I enjoyed the dynamic between Lena and Darius- there was a bit of an Insta-love situation, which can be tough trope for me to swallow. But they had some beautiful chemistry and banter, so it ended up all working for me. Still a lot of answers are left to be uncovered regarding Lena, which I’m sure will be addressed in the next book. Looking forward to seeing where everything goes, especially after the cliffhanger ending. 💀
All in all, an absolutely solid debut, and I am fully invested in this story. This author is definitely gonna make her mark in this genre.
✨Morally grey MMC
✨Strong FMC
✨Banter and humor
✨Touch her and 💀
✨Found family
✨Shifters & fae
✨Unique magic system
✨Dual POV
🌶️Slow burn that leads to spice (1.5)
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Reviewed in the United States on April 9, 2024
★★★★★ 4
Great Start to a new Series
Format: Kindle
I whipped right through book #1, and definitely finished the book eager for more. The main and supporting characters are all intriguing and well fleshed out. The humor, romance and action are all really well balanced. The one challenge I did feel the middle dragged a bit, a little repetitive with lots of wanting and talking about things but nothing happened until the end basically, but either way, I really enjoyed it!
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Reviewed in the United States on April 17, 2024
★★★★★ 5
One of the best debut novels I’ve read!
Format: Kindle
First, congratulations to Ella Dawes on an amazing book! You knocked it out of the park!
If you are looking for an action packed, 🌶️ 🌶️🌶️ story, this is the book for you.
Darius 🤤, the surly captain of the guard and an alpha at heart is to die for, battle scars and traumatic past and all.
Lena, is the fierce and strong FMC we all want to be. She’s string willed, without the needless stubbornness.
Fate has brought them together, whether they want things this way or not. The chemistry burning between Lena and Darius is undeniable, but can they set their differences aside and trust each other long enough to work together?
We’ve established the main characters are phenomenal but can we focus on the side characters for a bit? Griffin and Kace were so well written. I felt ai could be friends with them in real life.
The story is well written, the pacing was phenomenal and at times my heart hurt for the characters as if I knew them. This author just getting started guys, I cannot wait to see where she goes from here and how she continues to develop this world.
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Reviewed in the United States on April 2, 2024
★★★★★ 5
Great listen!
Format: Audiobook
I really enjoyed this audio book. Lead male character was a little too abusive for my liking, and that made me not root for the love match. But the story was interesting, and the voice actors did an amazing job.
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Reviewed in the United States on April 17, 2025