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Description
Rat α-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. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. 5. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 6. 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 an alpha-klotho protein (α-klotho) capture antibody. 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 alpha-klotho protein (α-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 Alpha-klotho protein ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | α-klotho (α-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 homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.5 ★★★★★
Based on 15 reviews
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Product Reviews
★★★★★ 5
Excellent Ice Maker
Color: Piano Black, Color: Piano Black
This 100% beats the heck out of the Frigidaire Gallery countertop nugget ice maker I had. The ice making is faster, the nugget quality is better, cleaning is easier AND it is waaaaay quieter than the other one. Let's start with the speed of the ice making. Within 15 minutes I have ice. Enough for at least a cup. The nugget: soft, easily crunchable, perfect sized just like that Sonic ice! Noise is basically non-existent compared to my previous machine. I don't even know this one is running most of the time unless I look over and see the display. Cleaning: It comes with packets for cleaning. Pour one in, set it to clean and in about 7 minutes it is done. I run mine with the same packet twice then drain via the easy to access drain plug in the bottom of the water well. Then I rinse twice and start it making ice again. I have an alarm set.for a reminder to clean it monthly, but I dont really need it because I can tell by the way the water looks in the well that it needs cleaning. Also, I always make sure to wipe down the sides of the well and wash the ice basket once monthly. I wish I hadn't wasted my money on the Frigidaire. Their warranty is a joke because it is no longer made by actual Frigidaire. I just tossed in in the trash. I didnt even get 3 months out of it before it started screaming and dying.
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Reviewed in the United States on May 20, 2026
★★★★★ 1
It runs a short period of time then stops with error codes.
Color: Piano Black
My husband had openings heart surgery his hospital stay was 9 days. During those 9 days, his only joy was the ice that the hospital had. So he asked me to order a machine that made that small crushed balls of ice. So I ordered this one.
It arrived nicely packaged.
It made wonderful ice. I only used water that was filtered with very good filters. Shortly after running it, I got an E1 error code. So I reported to the manual. There was no code listing for exact issues. So I chose to clean it ( whi h I thought was premature but had to try something). I followed the instructions on cleaning. It started to work again. Worked for another short wile then got the same code. I cleaned it 3x to find the same error code.
So I returned it and was awarded a full refund.
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Reviewed in the United States on December 1, 2025
★★★★★ 5
Perfect Little Nugget Ice!
Color: Piano Black, Color: Piano Black
I'm not one to usually do reviews, but I really love this little countertop ice maker. I was spoiled when I had an icemaker in my fridge at our previous house. This Countertop Ice Maker Machine is AMAZING and I'm so thankful for it! The size and shape of the ice is just right. It's super! The little nuggets are just the right size. They are not too hard. The ice becomes light and chewy. It's like I was eating ice skittles, lol. So simple to use. Fast too! I believe I can get a full tray (about 5-6 scoops) in less than an hour, maybe 45 minutes? I guess it makes some noise, it's not loud enough to bother me one way or the other. One button does it all - one quick tap to make ice and one short push to clean the machine. Simple to clean, cycle takes 15 minutes, dump the water (pull to edge of counter and open plug on bottom, let drain in small jug or bowl. Then, rinse, repeat twice and let air dry overnight and it's ready to go and go and go again the next day. It really feels sturdy and solid. I'm thinking if the inside is built as good as the outside this will last a long time. I've been seeing Typhur products more often and I know they are a quality brand name.
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Reviewed in the United States on October 25, 2025
★★★★★ 5
Built for people who love nugget ice!
Color: Piano Black
If you love nugget ice, this machine delivers exactly what you’re hoping for - soft, chewable, restaurant-quality pellet ice at home, consistently and quickly.
The biggest differentiator is speed and volume. It produces up to 35 lbs per day, and the first batch is ready fast. The ice texture is true crushed pebble/nugget style - not hard cubes disguised as “pellet” ice. It’s ideal for iced coffee, sparkling water, cocktails, and entertaining.
The stainless steel exterior feels premium and modern on the countertop.
Noise level is typical for a countertop ice maker, noticeable but not disruptive. It’s not silent, but it’s absolutely reasonable for the output.
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Reviewed in the United States on February 16, 2026
★★★★★ 5
Great Ice Machine
Color: Piano Black
This ice machine works amazing and the ice is perfect! Doesn't take long to produce the ice either! Would buy again!!
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Reviewed in the United States on May 25, 2026
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