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Description
Rat TRPM4 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 capture antibody against Transient Receptor Potential Cation Channel Subfamily M, Member 4 (TRPM4). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of Transient Receptor Potential Cation Channel Subfamily M, Member 4 (TRPM4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Transient Receptor Potential Cation Channel Subfamily M, Member 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Transforming receptor potential cation channel subfamily M member 4 (TRPM4), also known as melastatin-4, is a protein encoded by the TRPM4 gene. It is a calcium-activated, nonselective ion channel that mediates the transport of monovalent cations across membranes, thereby depolarizing them. Consequently, it plays a central role in luminal muscle cells, entorhinal cortical neurons, dorsal root and nasal neurons of the volar gland, endocrine pancreatic cells, renal epithelial cells, and cochlear hair cells. It participates in T-cell activation by regulating Ca(2+) oscillations following T-lymphocyte activation, which is required for NFAT-dependent IL-2 production. It also participates in myogenic contraction of cerebral arteries and controls insulin secretion in pancreatic β-cells. It may also participate in pacemaking or induce irregular electrical activity under conditions of Ca(2+) overload. It influences T helper 1 and T helper 2 cell viability and cytokine production through differential regulation of calcium signaling and NFATc1 localization. It enhances cell proliferation by upregulating the β-catenin signaling pathway. It also plays a role in keratinocyte differentiation. Its activity increases with increasing intracellular calcium concentration, but the channel does not transport calcium. | |||||||||||||||||||||||||||||||||
| 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 11 reviews
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Product Reviews
★★★★★ 3
Adult Fiction disguised as YA
Format: Kindle
When you first read the blurb and or the initial chapters of Crows, you’d think this was an adult fantasy book. The book even reads like adult fantasy, until you discover it’s anything but.
I think the biggest plot twist to this is in the way the author matter-of-factly tells you the exact age of her characters and its always coupled after explaining that characters extraordinary background.
Somehow it’s possible to be a seasoned assassin, rogue or racketeer (or all three, in that order) at just 13-17 years old.
Somehow you can live the life of a jaded 50-year-old at the ripe ol’ age of 12.
Somehow all the adults are either too rich, too dense or too dead (or all three, in that order) to operate a lucrative underground business, but kids are exceptionably capable.
Somehow it’s possible to be a father-figure to people the same age as you.
I’m specifically talking about Kaz here. He’s like that hardnose uncle who grunts all the time and tells you only what he wants you to hear. I loled each time he scolded a member of his crew.
Initially (before I knew the ages of these people) I thought Kaz & Inej’s relationship was more a teacher/mentor relationship, but then it morphed into this awkward romance where Kaz has less personality than a rubber ducky and Inej a lukewarm glass of water. To be fair, Inej got better as the story progressed, becoming the only one I really liked.
To the author’s credit, she does make a point to explain Numbuh One—I mean, Kaz’s abnormal cynical mannerism. But then the other characters (who are younger btw) act similarly. What’s their excuse? It was jarring to get behind, but what aided me through was that I just pictured everyone looking like the main cast from Kids Next Door and it helped tremendously!
Matthidus and Nina are virtually the same character. Their chapters were a drag on the narrative due to the fact that these two NEVER STFU about what they did to one another. It’s the “woe, it’s me” amped up to a thousand.
Jesper and Wylan were cool… that’s all I have to say about them.
Never at one point did I have a liking to any of the characters (besides Inej), neither did I feel they were ever in any real danger. Kaz is a walking-talking dues ex machina. The characters find themselves in a bind and he already has the solution to their problems. He’s like Superman morphed with Batman while the other characters are just your bog standard Metropolis denizens who constantly need saving or guidance.
In a nutshell, he’s a Mary Sue.
I found the pacing bogged down to the point that it was hard to keep my eyes open while reading. Not because it’s badly written but because the author constantly reverts back to the characters’ backstory over and over again. There would be a needless flashback in the middle of a fight scene. Characters near death randomly float off to dream land while getting the crap beat out of them. It was jarring.
Closing remarks
All-in-All, Crows is a unique book. I can’t really talk about the plot without spoiling anything, but it’s innovative and engaging. I can say the book is worth your time if you can circumvent the kids playing grownup, then you should enjoy it.
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Reviewed in the United States on March 28, 2019
★★★★★ 5
Good start to a series
Format: Kindle
I delayed reading the series for reasons I don’t remember. But my TBR list is huge so I thought I’d take a shot of this and I was pleasantly surprised. I didn’t think the blurb about it was anything special. But it was a very good book. It took some interesting twists and turns. I am so glad the second book is already out. Because I would not have waited patiently. Very slow burn but good storyline. 🔥🔥/5
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on January 3, 2025
★★★★★ 4
A good read
Format: Kindle
Multiple points of view. 3 Alpha men and an Omega male. She is a Beta in training for a new program placing betas in Alpha/Omega packs. Mila is only doing the program for the money to take care of her dad. She wasn't expecting to fall for a pack but when she sees this packs Omega she is done for. There is just something about him. His Alphas are good looking as well. Too bad she is hiding a secret and their government is acting shady. I liked it and can't wait to see where their story goes.
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Reviewed in the United States on November 14, 2023
★★★★★ 3
Slightly repetitive but I did love some things
Format: Kindle
I love this type of story. And omegaverse is one of my all time favorite genres. But there are a few things that pulled me out of my enjoyment while I was reading. It was repetitive at times as well as struggled with telling not showing. So we didn’t always feel like we were experiencing things with the main character. There were also some plot holes but they may still be answered in part 2.
Now this isn’t to be said I didn’t enjoy parts of the story. I loved the almost instant love between Mila and Oliver. And how he started changing around her.
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Reviewed in the United States on February 15, 2024
★★★★★ 5
delightful read
Format: Kindle
What a delightful read. The characters are awesome, the plot was so good, I loved it. I was intrigued and it kept me wanting more. Told in multiple pov, the book sucks you in and doesn’t let go. I cannot wait to read the next book.
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Reviewed in the United States on January 30, 2025
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