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Description
Rat NPHN 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: Urine 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
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: Urine 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 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 utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP enzyme conjugate are sequentially added to microwells pre-coated with a nephrin (NPHN) 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 nephrin (NPHN) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Nephrin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Nephrotic hormone (NPHN) is a protein essential for the proper functioning of the kidney's filtration barrier, which is composed of slit endothelial cells, the glomerular basement membrane, and the epithelial capsule. It is a transmembrane protein and a structural component of the slit diaphragm. It is present in an intricate network at the apical end of the capsule cells, imparting a strong negative charge that repels proteins from passing through Bowman's space. Defects in its gene, NPHS1, are associated with Finnish congenital nephrotic syndrome, resulting in the leakage of large amounts of protein into the urine, a condition known as proteinuria. It is also essential for cardiovascular development. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Urine, tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.5 ★★★★★
Based on 21 reviews
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Product Reviews
★★★★★ 5
Monstrously Sweet
Format: Kindle
This book packs an emotional punch, especially if you’ve experienced childhood trauma (particularly abuse and/or neglect). Alexandra creates these three strong protectors who love her and will do anything to make her feel happy and safe. She started out drawing them when she was a small child, and then transitioned to stories as she grew up. Lucien, Elwin, and Kylo look frightening in their monster forms, but it’s clear that they would never hurt Alexandra, and she has never felt afraid of them. They have very distinct personalities that we get to know better throughout the book. The love and devotion they have for Alexandra is sweet and unwavering.
This is a fantastic and creative story! The world-building is wonderful - it’s a shared world with M. Sinclair, and takes place at a university for supernatural beings. The additional world-building within Alexandra’s story added even more depth and intrigue. The spicy times were 🥵 and it’s more of a medium burn, but it works with the characters and I expect that it’ll pick up quite a bit in the next books.
Monsters Within has a unique way of addressing mental health issues that can come from being abused and neglected. There are some flashbacks to Alexandra’s childhood that provide some insight but they don’t go into heavy details.
I absolutely adored this book and can’t wait for more!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 21, 2022
★★★★★ 4
One of us
Format: Kindle
Alexandra is literally one of us. She's just lucky her fictional men became a reality hahaha. This was a good start to what I know is going to be a great series. This was fast paced and kept me entertained. Im ready for the chaos to slow down a little bit so we can get more into the character development. There's so much happening, I feel like we hardly know anything about anyone BUT that's why this is a series so let's get to the next one!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 19, 2025
★★★★★ 5
Love this!
Format: Kindle
So far I'm loving this series! The FMC is tough and sweet and I love her MMCs. Its a very interesting story so far. On to book 2!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 27, 2026
★★★★★ 3
Alex and her monsters
Format: Kindle
A somewhat intriguing story that did drag a little despite giving us some world building. The explanations just took too long even if we don’t get the full picture yet. I will say the story does have some humor and is very hot. I may pick up the second book at some point.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 11, 2023
★★★★★ 5
Interesting Monster Academy
Format: Kindle
Alexandra is tired of being alone, using writing to escape from her reality. But when her monster protectors in her writing come to life, her life is going to become a little more exciting than she ever anticipated.
This was a fun take on an academy romance. We have two different realities, one for the real world and one for the supernatural world. I loved the Dark Imaginarium Academy and the intrigue it held throughout the story. The plot was fun and the characters were written beautifully. Alexandra, Elwin, Kylo and Lucien were great characters and have the best out of every relationship dynamic. My heart does belong to Lucien, because who doesn't love a murderous psycho willing to kill anyone who hurts you?
I wish there was a little more intrigue to the plot itself. The story was fun to read and well written, but it's still a straightforward Academy romance. I would've liked to see something that made the story really stand out more.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 21, 2022
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