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Description
Mouse ATP1b4 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 ultrasonically disrupted or repeatedly frozen and thawed. Finally, the homogenate is centrifuged at 5000×g for 5-10 minutes and the supernatant is collected 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 utilizes 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 an ATPase, Na+/K+ Transporting Beta 4 Polypeptide (ATP1b4) capture antibody. After incubation and washing, the sample 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 amount of ATPase, Na+/K+ Transporting Beta 4 Polypeptide (ATP1b4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse ATPase, Na+/K+ Transporting Beta 4 Polypeptide ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | The protein ATP1B4 (ATP1b4), also known as ATPase Na+/K+ transporting family member β4, is encoded by the ATP1b4 gene. It functions as a transcriptional regulator during muscle development through interaction with SNW1. It has lost its ancestral function as the β subunit of the Na,K-ATPase. Diseases associated with it include mannoses, Beta A, lysosomal, and thyrotoxic periodic paralysis. Pathways involved include GPCR signaling and the transcriptional activity of the SMAD2/SMAD3-SMAD4 heterotrimer. Two transcript variants encoding distinct isoforms have been identified for this gene. | |||||||||||||||||||||||||||||||||
| 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 and other biological fluids |
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4.3 ★★★★★
Based on 773 reviews
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Product Reviews
★★★★★ 4
Happy Wanderer
Format: Flexibound
City Trails is not a guided walking tour (like the Freedom Trail here in Boston) of the Metro DC area. No addresses or street names are noted in the blurbs. To actually visit any of these places, you’ll have to consult a real map. For instance, the chapter “Statue City” highlights notable statuary around town. But the Capitol Building statues (in SE DC) are far from the Cathedral ones (in NW DC.) The themed groupings (G-G-G-Ghosts, Animals Around Town, Water World and more) are less maps to any place and more of an interesting overview of our Nation’s amazingly diverse and action-packed city. It’s best read as a primer on experiencing the flavor of the city (I lived and worked there.) It reads more along the lines of the “Weird But True” series made famous by National Geographic for Kids.
I don’t see this being of value to tourists in town for a limited time whose sightseeing is going to include major attractions like government buildings (White House, Capitol), museums (Smithsonian), some monuments (Jefferson, Lincoln, Washington) and other popular sites (Ford’s Theater.) This guide is actually best suited for the Metro-area (WDC, MD and VA) resident – child or adult - who wants a deeper dive into their hometown’s off-the-beaten-path sights and stories. A well designed and written book of historical trivia.
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Reviewed in the United States on August 29, 2018
★★★★★ 4
Nice way to learn about a trip to D.C.
Format: Flexibound
I got this for my kids to read before we went to Washington D.C. The pages are colorful, illustrated, and have short bursts of interesting details about the various attractions available to tourists who are visiting. My kids were eager to find the places on our itinerary and read about them ahead of time. They learned what to expect and were sure not to miss the important aspects of our tours. This book is recommended for 9 to 12 year olds and I think that is the perfect range. There is just enough information to peak their interest and not so much that they get bored by reading a bunch of text. The Table of Contents wasn't that informative in finding specific places, but the index was. My kids preferred to leaf through the whole book and find what was interesting to them.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 28, 2018
★★★★★ 5
Learning while having fun
Format: Flexibound
Great book for the grandchildren - and the parents enjoyed it with them
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Reviewed in the United States on January 20, 2018
★★★★★ 3
Not good for travel, picture book only
Format: Flexibound
This is a beautiful book with fantastic illustrations and an engaging color palette. It includes a good variety of historical background information and sightseeing locations. However, it is a better picture book for browsing only. It is not a good travel book for kids to plan their own adventures. Who has ever heard of a travel book without maps?! No maps, no directions, no coordinating subway/bus maps. The printing is exceptionally small, almost too small to read. The book should have been made larger to accommodate the text. The text is excellent, but it printed as if the publisher never expected the kid/parent to read it. On one hand, our family really enjoyed how engaging the book appeared, but we were disappointed in its quality as a travel book. It shouldn’t be marketed as a travel book, but a geography book series.
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Reviewed in the United States on February 1, 2020
★★★★★ 5
Details
Format: Paperback
This book is easy to read and provides the details you need to know to have a great trip.
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Reviewed in the United States on May 6, 2023