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Description
Human NCCT 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-chloride symporter (NCCT) 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-chloride symporter (NCCT) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human sodium-chloride symporter ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | The sodium-chloride cotransporter, also known as NCCT, is encoded by the SLC12A3 gene located at 16q13. It is a cotransporter in the kidney that reabsorbs sodium and chloride ions from the renal tubular fluid into the distal convoluted tubule cells of the kidney. It is a member of the SLC12 family of electroneutrally coupled chloride cotransporters. Loss of its function causes Gitelman syndrome, an autosomal recessive disorder characterized by salt wasting and hypotension, hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. | |||||||||||||||||||||||||||||||||
| 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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Product Reviews
★★★★★ 5
Fav Chew Toy 🦴
Color: Blue, Color: Blue
My baby is half rottweiler and half doberman &’ he has a pretty strong grip when it comes to chew toys. But this one, it’s his favorite toy! He never leaves the house without it. It’s also really fun to add his favorite treats in there when’s he bored to keep him entertained! Not loud at all and will keep him from chewing on other things and not heavy as other rough chew toys!
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Reviewed in the United States on May 31, 2026
★★★★★ 5
Great you made super well
Color: Blue Lil Snoop, Color: Blue Lil Snoop
This is such an awesome toy. It’s super durable strong bounces and a perfect size. We got the small. We got blue because our pup can see it. It allows for creativity and endless limits to what you can make for it to be a fun experience. It has a nice sized hole in the center which allows you to add treats and food. Pop it inward and it slightly if large enough prevents the treats from coming out super fast. The ball rolls bounces and they can grip it nicely.
It is easy to clean
The only thing I didn’t like was the way the plastic tabs were situated inside the toy as you just be careful you don’t allow it to drip inside and dog gets it. No noise just fun. Our 15lb Cavapoo loves it
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Reviewed in the United States on February 17, 2026
★★★★★ 3
No challenge once they figure it out once, or if they have a long nose
Color: Blue
The first time I used this with my 9 month old Shepard mix, it was great. We used about 1/3 cup of her kibble and added the Nook to make it harder. It kept her working for 20-30 minutes. However, now that she knows how to remove the Nook, and stick her snout into the opening and lick up the kibble, it lasts about 5 minutes, and is no longer a challenge. She still enjoys playing with this, even when it is empty, and hasn't it destroyed it yet, so for that 3 stars.
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Reviewed in the United States on April 5, 2026
★★★★★ 5
Buy it — it’ll be your dogs best friend!
Color: Orange
Best treat toy ever! In the beginning of 2025 I bought this for my 12yr old Alaskan Malamute who loves puzzles and brain games. She loved it and played with it every day. Sadly, she passed middle of this year. Now in December, I have purchased this toy again for my newly adopted 10 month Great Dane puppy. Again, it’s an absolute hit! My Dane eats very fast, so I actually use this as a way to break up her meals. I put about half her meal (freeze dried raw kibble) in her slow feeder bowl and fill this toy about two times to feed her the other half. Also, we are crate training and before I leave the house I put treats in this toy and leave it in the crate with her. She has bad separation anxiety, but this toy actually seems to help calm her down because she enjoys it so much. I have bought many treat toys — this one is supreme. It’s not too loud or too difficult for the dogs to play with. So far it has held up without being chewed apart. It’s good quality, great size (orange for large breeds) and honestly, worth every penny.
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Reviewed in the United States on December 12, 2025
★★★★★ 5
Well made dog toy
Color: Blue
The snoops are great for our active labrador. He is a very aggressive chewer, and so far, has not destroyed a snoop yet. He has mulitples. They are a great size for filling with treats or kibble and they keep him engaged in the activity. He even likes to carry them when he does his zoomies sometimes. They are lightweight and sturdy, and the material is nice and flexible. Great toy / puzzle ball to have around for your pup!
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Reviewed in the United States on June 9, 2026