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Description
Rat NHE3 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 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 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 sodium–hydrogen antiporter 3 (NHE3) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the sodium–hydrogen antiporter 3 (NHE3) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Sodium–hydrogen antiporter 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Sodium/hydrogen exchanger 3 (NHE3), also known as sodium-hydrogen transporter 3 or solute carrier family 9 member 3 (SLC9A3), is a protein encoded by the SLC9A3 gene. It is a sodium-hydrogen antiporter. It is found in the apical region of proximal tubular epithelial cells of the kidney, in the apical region of enterocytes, and in the basolateral region of duodenal and pancreatic cells, responsible for releasing HCO₃ into the duodenal lumen. It is primarily responsible for maintaining sodium homeostasis and is also indirectly involved in buffering blood pH. | |||||||||||||||||||||||||||||||||
| 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 | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.7 ★★★★★
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Product Reviews
★★★★★ 5
This is really a great read for individuals wanting to increase their knowledge of ...
There was so much information in this book that has never been bought to light. This is really a great read for individuals wanting to increase their knowledge of the Black Power Movement.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 20, 2016
★★★★★ 5
Review of Deacons for Defense
Format: Paperback
I found this book to be extremely informative about the Deacons. I was in Monroe ,LA in 1965 as a staff lawyer for the Lawyers Constitutional Defense Committee. We represented Deacons in various instances. This history filled in innumerable gaps in my knowledge of the Deacons. To the extent of my recollection the facts and analysis seemed accurate to me. This book is recommended for anyone with an interest in acquiring a full understanding of important civil rights organizations which contributed to the Movement in the '60's'
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 3, 2014
★★★★★ 5
I love this book
Format: Paperback
I love this book. Some of the incidents took place in my home town of Jonesboro, LA. I knew many of the men (and women) who risked their lives to take a stand for racial equality. CORE's Jonesboro Freedom House (although it is vacant) still stands today, and its structure has not changed. This house, along with many of the pictures of individuals, are shown between pages 107 & 108 of the book. I shared this information with some of my students. They were quite surprised that our small town was a part of History!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 14, 2017
★★★★★ 5
Funny, Interesting, Informative!
Format: Paperback
I hiked the PCT in 2016 and I wish this had been available then. There are a lot of misconceptions about the desert section of the PCT (first 700ish miles) being flat, monotonous, and unpleasant. Even once you are out on the trail hiking through varied terrain, it is hard to shake off the "desert mentality" that comes from listening to those misconceptions. One of the reasons I love this book is because of how well the author paints an accurate picture of the beauty and constantly changing scenery the desert has to offer.
All the information you would expect from a guide book is here, including mileage, water sources, elevation, etc. There are also a TON of photos that will make you question why you spend anytime indoors. I mean this literally. This book is worth buying for pictures of the Sierra sections alone. The damn near turn by turn navigation descriptions of each section are also a good reason for your purchase.
The author is funny. I wanted to say that in a funny way but she's funny enough so if you want to chuckle, wait for the book. Her passion for the PCT seeps through her writing and it is impossible not to feel equally enthusiastic. On a multi-day (or multi-month) hike, that enthusiasm is almost as important as your next water source...except not..but ALMOST.
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Reviewed in the United States on December 18, 2017
★★★★★ 5
Anyone Can Do It!
Format: Paperback
My husband and I are hiking the PCT and this book has come in very helpful. We especially appreciate the areas we can park at the beginning and end of each section. It is making the trail very doable for two older people who could never make it otherwise. I tear out the section, make a copy and highlight some of the details for example a gate at 4.5 miles. It helps encourage us to continue when we know about how far we have left to go. Also the little map showing elevation gain and lost. The last 17 mile trip we did last week was predominantly up hill. Knowing that ahead of time prepares us mentally for a difficult hike. Our next section is 10 miles downhill so we will do that as a day hike and bring the dog. For anyone who thinks they can't do the PCT this is the perfect book. If I can do it Anyone Can Do It!
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Reviewed in the United States on February 14, 2022
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