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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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4.0 ★★★★★
Based on 23 reviews
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Product Reviews
★★★★★ 5
solid no nonsense watch
Color: Black/Yellow
This watch has many key features I needed in an everyday watch.
- 5 separate alarms which you can set to a single day of week, weekend, weekday, or all week. useful for let's say trash day reminders or school day pickup reminders. The flexibility of the alarms is really key.
- 9 interval countdown timers (repeats when done)
- 1 countdown timer
- stopwatch100 lap (more than enough)
- indigo light, excellent nighttime illumination
- excellent water resistance and durability. i've had mine for nearly 2 years and it still looks sharp. some of the lettering on the front has rubbed off, but most still visible, colors hold well. and i've put mine thru all sorts of abuse over the years.
- large screen and digits for easy viewing at work. i have to record times at work so this was key. date and day of week is easily visible as well.
- good battery life. if you don't use light a lot but use alarms regularly can last easily between 1-2 years
- excellent price for about 30$
CONS:
- no vibration, but i don't really need this
- dual time zone... actually not ideal because you switch by pushing & holding the start/split button below the face. i accidentally seem to push and hold this button a lot thus it switches my timezone. i've gotten around this by just setting both times to be identical but a bit of a pain
- resin band is durable but as always the little moving strap part to tuck away extra band breaks after a while. I discovered a neat trick to use my daughter's small hairband tie folded over twice to secure the extra band now. durable and doesn't look bad (if you pick the right color).
- pain in the butt to change the battery (uses CR 2025 by the way). you have to remove both resin band parts first as it slightly overlaps the back case. then there are 4 tiny screws. once the bands are taken off it's pretty straight forward. make sure to align the thin rubber seal before you put the cover on. mine leaked water in after a change until I realized the seal was not sitting properly.
Overall, I've been thru smartwatches, fancy watches, solar watches, ... mainly many casio and timex watches. this is by far one of the most practical and best watches I have. I'll be curious to see what google/apple create in the smartwatch category of the years.
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Reviewed in the United States on September 6, 2013
★★★★★ 5
Great sports/fitness watch
Color: Black/Yellow
I will start my review with an admission:
I hate digital watches.
I think 7-segment numbers look ugly, and I prefer the ability to see the 'distance' between times visually that you get with an analog face.
But I needed the timer and lap counter for my job, and I have always wanted to do interval training to improve my speed and endurance while cycling. This watch had everything I needed and more. I was aware that Timex was a respected brand, and the T5E231 was the only watch in my price range with the ability to count 199 laps.
I have to say, though, that after a week of use, I actually love this watch. Not only does it meet my needs functionally, but it actually brings me pleasure to wear it and operate it. The yellow trim around the inside of the bezel clearly identifies it as a sport watch, yet it seems to fit in well in formal settings. The interface is incredibly user-friendly and satisfying to operate. I never thought I would refer to a watch's "interface", but this watch actually has one. Pressing most of the buttons results in actual text being displayed on the screen telling you what the button did, or what will happen if you hold that button down just a little longer. In most modes there are even little labels that appear on the screen by the buttons telling you what they do in that mode. Figuring out how to use almost all of the functions only required five minutes of random experimentation. The only thing I had to consult the manual about was how to switch the chronograph into lap counter mode. I just wish there instructions explaining how to fold the manual back into a size that fits inside the watch stand.
There are so many little touches that make it clear the designers at Timex really take pride in their work. Here are my favorites:
-When turning night mode on, the Indiglo lingers for another few seconds after releasing the button. But when turning it off, the light extinguishes immediately! It should, since if you are turning night mode off, it's probably daytime and you don't need the Indiglo anymore.
-Being able to scroll numbers backwards and forwards while setting times. On other watches it is so frustrating to 'miss' the number you want and have to press the button 60 more times.
-Little icons appear on the home screen letting you know if the timer, chronograph, or alarm is running in the background. Most watches only have an icon for the alarm.
-The speaker plays a different sound for the alarm and the timer, so you know which one is going off without looking at the screen. Also the Indiglo light flashes.
-The Indiglo system is clearly a masterwork of engineering. It looks evenly lit and is very easy to read, yet it doesn't illuminate anything other than the display (unlike the backlight on a phone, for example). This must save a lot of battery power, since lighting up other objects around the watch is a waste of electricity. Besides, you have your phone for that!
-In timer mode, you can see what time the timer was originally set for on another line below the countdown.
-The AM and PM appear in the same place on the display; there is just one little segment that lights up to turn the P into an A.
-Many of the buttons that perform an irreversible or potentially unwanted function (such as resetting the chrono or clearing workout data) require being held down for several seconds so you don't trigger them accidentally.
-The watch tells you how much memory is free for storing workouts.
There is only one problematic thing about this watch (and it may actually be a problematic thing about myself). In order to activate the FLIX system I am required to wrench my arm so hard I nearly dislocate it. It is painful, and takes way more effort than just pressing the Indiglo button. This doesn't bother me, because I didn't buy this watch with the intention to ever use FLIX, but it is somewhat frustrating that it is so hard to use. However, it is possible that my technique is flawed, and there is an easier movement that will activate it. But I don't really want to experiment; I like my arm in its current uninjured state.
In all, I am very satisfied with my purchase. I love everything it does, and the only 'negative' probably says more about me than it does about the watch. Considering my abhorrence of digital watches, I am surprised that I like this one so much. I don't plan to wear it any other time than while at work or working out, but for those times it is not only tolerable, but actually enjoyable.
I highly recommend this watch!
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Reviewed in the United States on February 19, 2013
★★★★★ 5
Great Buy
Color: Black/Green
Very nice watch. Simple design and I love the luminous face .
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Reviewed in the United States on May 21, 2026
★★★★★ 5
Decent watch for the price and very easy to read
Color: Black/Blue, Color: Black/Blue
I would consider this equal to the Timex Easy Reader except without Indiglo and less expensive. It is a nice size with quoted average water resistance. It has a press-down back and mine has a gunmetal color sort of sand blasted finish case. I don't know if it's coated or the actual metal color. The strap which came with it is of the NATO type and of decent quality. In the pic it is wearing the rubber strap from my Vaer watch which is a quick release rubber one. NATO straps are fine if you don't mind the double thickness of strap between the watch back and your skin. The spring pins aren't the typical ones which have the raised parts which allow you to use a removal tool. They are quite difficult to remove but I did it using a box cutter and by applying pressure I was able to eventually work them out and put normal ones on. The strap width is 20mm. I have a bunch of other straps which I can use with this watch. The full face lume is quite bright but I don't know how long it lasts just yet. If in a couple of years it needs a new battery, don't use a kitchen knife to snap off the back, use the proper tool which you can find on Amazon and follow the online tutorials on how to get it back on. All in all it was a good purchase.
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Reviewed in the United States on December 16, 2023
★★★★★ 4
Good watch
Color: Silver/Green
This actually a good watch for the price. It glows in the dark due to solar exposure during the day. The wrist band was a little long for my first.
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Reviewed in the United States on May 25, 2026
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