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Description
Rat sα-klotho ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 3. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. 4. 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. Preparation of standard gradient working solution: Add 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 2000pg/mL). Then dilute to the following concentrations: 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 2000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 1000pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used 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 soluble alpha-klotho protein (sα-klotho). 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 soluble alpha-klotho protein (sα-klotho) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Soluble alpha-klotho protein ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | α-klotho (sα-klotho) is a transmembrane protein that plays a role in controlling insulin sensitivity and appears to be involved in aging. It alters cellular calcium homeostasis by increasing TRPV5 expression and activity (reducing renal phosphate reabsorption) and decreasing TRPC6 expression and activity (reducing intestinal phosphate absorption). It can inhibit oxidative stress and inflammation, thereby reducing endothelial dysfunction and atherosclerosis. Aerobic exercise increases plasma α-klotho levels, thereby reducing endothelial dysfunction. | |||||||||||||||||||||||||||||||||
| 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 | 31.2-2000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates and other biological fluids |
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4.6 ★★★★★
Based on 21 reviews
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Product Reviews
★★★★★ 4
book 2
Format: Kindle
This was a great read. I loved both main characters!!
It has spice, sass, the sweetest FMC, rich family backstories, and a HEA!
Hockey, romance, and hotness. What else could you ask for?! Give it a go!!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 24, 2026
★★★★★ 3
Steamy with Some Good Character Development, But a Little Cliche and Unbelievable
Format: Kindle
This book tells the story of Grace and (Johnny) Logan, a couple who hook up right away and then fall in love as Logan has to make up for the fact that he initially rejects the idea of getting more serious with Grace. The plot is pretty flimsy, although there is decent development of outside conflict, including Logan's alcoholic father and his struggles with his potential hockey career. There are some very steamy scenes--lots of them, actually-- and some cute moments, but in the end, this isn't a particularly memorable story.
To be honest, I have a love/hate relationship with this series (I read the first one, too). On one hand, I think Elle Kennedy is a good writer with a flair for dialogue who knows how to script a steamy love scene. She also does a solid job for the most part in fleshing out her main characters, even if supporting characters often seem like walking cliches ("The Overprotective Dad" vs "Alcoholic Dad"; "Rebellious Best Friend" vs. "Straight-Laced Best Friend"). There are a lot of genuinely funny lines, too, even if they're sometimes of the stereotypical frat boy variety (as a lot of the characters are stereotypical frat boys).
Speaking of stereotypical frat boys, this dovetails into what I hate about this series. I have to admit to not being a fan of the Greek system in general so that may be a bias that's affecting my reading experience, but there's something over-the-top and a little disconcerting about the way Kennedy depicts both the sex-fueled parties and the conversations between the guys about their sex lives. I'm not saying her portrayal of Greek life is entirely unrealistic, but the scenes have a cliched, obvious feel, and although she means to set a sexy tone, there's something icky about the way these guys get whatever they want, whenever they want it (and it's not just sex on demand, but apparently amazing sex with hot women on demand). All the guys are gorgeous horn dogs who get drunk but never seem to be anything less than smoking hot and cool. Oh, and apparently, all this partying doesn't negatively impact their training regimen). Most of the girls throw themselves at them, either alone or in pairs. Plus, in both books, I had some trouble believing that the heroines really hung out at frat parties and knew the athletes by name. Both Hannah and Grace (and even Ramona, for that matter) struck me as young women more apt to avoid frat parties and maybe hang with the multitude of other non-Greek members who populate a college campus and host off-campus parties. (To that end, how about a hook-up between one of these athletes/frat boys and a young women who has no interest in an athlete/frat boys? I went to a big school, and at least two thirds of the campus probably couldn't identify one athlete by name.)
Ultimately, I enjoyed aspects of this book and liked the main characters by the end of the novel (Logan wasn't that appealing for about half of the story, but I warmed to him eventually). And it definitely is more expertly plotted and well written than a lot of other NA novels out there. But if this series is to continue, I'd like to see the formula shaken up.
Grade: C+
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Reviewed in the United States on May 23, 2016
★★★★★ 5
HOT AND HEAVY!
Format: Kindle
Awesome read! It’s was funny, sexy, dramatic, endearing. Great story and plot line. Loved it! About to read the score now.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 31, 2026
★★★★★ 4
I finally took the plunge. A satisfying star read
Format: Kindle
What a chicken I was to leave off reading this book for YEARS because I couldn’t cope with Logan’s “situation” (no spoilers!) & how it could possibly be sorted in his own book. Oh ye of little faith! Elle Kennedy isn’t a #1 author by CHANCE! Oh no, that is TALENT baby! She took the end of book one, ran with it, then flipped that mother sucker on its head to dig deeper into Logan and learn about Grace. These two were definitely made for each other.
The story spans from one school year to the next and we get lots of context too. There are some side characters that I’m not sure o know why they were added as they didn’t add to the story and a particular side character that really needed an ending as she just disappeared. Why was she so unhappy? What was she getting into off page? Was she ever forgiven?
The s story seemed to end abruptly, and that threw me off a bit. I kept going like I was gonna flip another page, and that was the end! Besides the fact of feeling like there are so many things left unsaid this was a good book. Definitely liked book one more but that could also be because the show just came out on prime but nahh, I loved to book one even a decade ago, and it’s still amazing to this day!
The hard part of this was the female narrator on the portions when I listened…Lorelei Avalon, she was NOT CAST properly in my opinion at all. She’s not a bad narrator, she’s just not suited, even remotely, for a late teens early 20-year-old college student. Her voice is more for characters late 40s early 50s in my opinion. I’m glad on book 3. It looks like we changed the narrator so I hope to mostly listen to that one. As always, Lee Samuels did a phenomenal job. He’s been in the game for a while, so maybe that’s what helps make it right for him. But he definitely knew how to change the nuances of his voice to suit whatever character he was reading at that time.
All in all, this was a really good book that I’m sad. I waited so long to read. Thanks to campus diaries coming out on TV? I’m ready to finally finish up this series! Book 3 here I come!!
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Reviewed in the United States on May 28, 2026
★★★★★ 5
🎧
Format: Paperback
The start of this book had me nervous but I ended up really loving this book! I loved how aware Logan was to what he was doing to Grace and stopped it even though it hurt to read. I loved that Grace made him WORK for her forgiveness and some parts were even SO funny. I loved all four of the guys and their great banter. My sweet baby Logan really was going through it in his personal life and his story resonated so much with me. I cried multiple times while reading and cried happy tears at the ending. It was perfect and exactly how I hoped it would end.
🎧 The narrators did a fantastic job with the dual narration.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 17, 2026
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