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Description
Human NADK 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 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves 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 NAD kinase (NADK) capture antibodies. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of NAD kinase (NADK) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human NAD Kinase ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | NAD kinase, also known as NADK, is an enzyme encoded by the NADK gene. It converts nicotinamide adenine dinucleotide (NAD⁺) to NADP⁺ by phosphorylating the NAD+ coenzyme. NADP+ is an essential coenzyme that is primarily reduced to NADPH via the pentose phosphate pathway, providing reducing power in biosynthetic processes such as fatty acid biosynthesis and nucleotide synthesis. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.6 ★★★★★
Based on 30 reviews
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Product Reviews
★★★★★ 5
Delightful little device now that I've had a practice run with it
Color: Silver
I just picked this up to make my own cappuccino and breve and I must say that I've fallen in love with the thing. My first use was a bit of a misfire owing to my inexperience with it, but the second time worked like a champ. The trick is to ensure that you have plenty of empty head space in the container to develop a good vortex and foam volume while still having a tall enough fluid column to keep the mixing head mostly submerged while generating the vortex.
My first attempt involved using about half a cup of half and half in a small saucepan. I had more than enough head space, but I was only barely able to keep the mixing head (it comes with two: a single turn coil and a 3-turn coil of coil) submerged and that went poorly, leaving me with a bit of a mess to clean up on my rangetop as the lack of fluid column ended up slinging the fluid around and out of the saucepan.
The second attempt went far better, as I transferred the barely-simmered half and half back into the Pyrex two-cup mixing cup in which i initially measured the 4 fl. oz. This time it worked beautifully. There was just enough unfoamed column height to keep the mixing head well covered prior to powering up and it just barely kept me sufficiently covered while powered up to maintain a very vigorous yet controlled vortex. This method also had the advantage of telling me exactly how much total foamed volume I had generated. The resulting breve had a wonderfully thick and frothy head in my mug and resulted in a delightfully topped beverage which, if anything, had a bit too much foam and a bit too little liquid. That's on me, however, since I wanted to get a feel for the device under better conditions to build my own experience; I'll just run the frother for less time next time.
Cleanup of the device itself is quite easy: simply detach the mixing head and shaft from the motor base and rinse. I always hand-wash my dishes, so I'm not in a good position to speak to its machine wash compatibility, but I suspect that it would come out just fine, provided that care is taken not to bend the shaft or coil head out of shape.
The device itself is small enough to fit in a purse and should travel quite nicely, though you'll need to commit to a single mixing head, which will almost certainly turn out to be the smaller one.
It's really nice to be able to make cappuccino/breve at home any time I see fit now. It's going to make a well-loved addition to my kitchen.
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Reviewed in the United States on December 6, 2018
★★★★★ 4
Worked well, fast, then overheated. Contacted Vomelon and they were quick to help me replace unit.
Color: Silver
Update ***Vomelon customer service is great! I contacted them about my issue, they responded quickly and worked to resolve it. They're giving me a new one! Hope this one works out. I'm adding back a star for their service.
Update 4/18/20 While making whipped Dalgona coffee today the unit slowed down the completely shut off. I pressed the power button again to restart and same thing, it starts at high speed then slows down and shuts off. I will be contacting seller. I’m taking away a star, can’t believe this is happening.
First day using this frother with the batteries provided and the body became very hot. Used it for about 3 minutes and the heat became uncomfortable in my hands. Took out the AAA alkaline batteries “Nai Li Wang” brand made in China and replaced them with some Kirkland AAA that I had on hand. Problem solved! No overheating with the new batteries. Throwing away the China made batteries. Would’ve been 5 stars if it weren’t for the overheating.
Other than that mishap, the frother was easy to use and pretty powerful. Whipped up my coffee with maple syrup (making Dalgona coffee) really quick. I like that it has a cover for storage. I don’t have much space so having a compact size with a cover is convenient, I can put it in a drawer or storage box without damage to the whisk.
Update: Day 2 of use
Body of unit became warm but not hot as with original batteries provided. I used new batteries. Had the unit on for 4-5 minutes to whip my dalgona coffee. Not sure if the unit is supposed to get warm? Is it normal? I will contact seller.
Besides that, my coffee got whipped pretty quick. I want to upload a photo but seems that Amazon has taken away that function for me? I could upload photos for my previous reviews on other products.
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Reviewed in the United States on April 16, 2020
★★★★★ 5
Great Little Frother.
Color: Silver, Color: Silver
I really like this this little frother. What I noticed first is that it comes with a cover for the frothing arm. That’s very practical if you travel with it. I have traveled with a frother before and I put it in a toothpaste box, not much protection there.
I use this 2 to 4 times a day. I can come pretty close to creating a Starbucks flat white at home. That saves $$$. The next thing I really like is that I can remove the arm for cleaning. If the arm does not come off clean up is a bit tricky.
How does it work? Great! It has one speed, Fast. The power button is on the base of the handle so I just had to adjust the way I hold the frother when I turn it on. One thing, make sure the froth head is all the way into the liquid when you start it, lol. This little machine whips up a thick froth fast.
It comes with 2 frothing heads, a three spring version as well as a standard one spring.
There is a lot of plastic, but it doesn’t look or feel cheap. Actually I think it helps keep the weight down. I travel some and I appreciate when things are light.
Over all, this is really nice it’s light portable and works great. I highly recommend this product.
I was given this product for review and I am so glad they choose to contact me. I have been frothing milk for my coffee for the last few years.
This has now became my go to frother.
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Reviewed in the United States on July 12, 2019
★★★★★ 5
Great Frother
Color: Silver
The best frother I’ve owned! Love that the whisk is removable for washing, the end is flat so it stands up on its own and it’s pretty powerful.
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Reviewed in the United States on February 20, 2026
★★★★★ 1
Almost right but connector sucks
Color: Silver
I much prefer a frother with swappable batteries and always use rechargeable AAA's and never disposable one's. The power button is in a comfortable spot as opposed to everyone else's odd and very uncomfortable side placement. Placing the power button on the side make you have to twist your entire arm to have your thumb facing down to be able to press anything whereas this one is right on top and can hold it like a simple joystick with no twisting of my arm.
However, these plastic attachment points crack waaay too easily. I've already bought 3 of these and this is the part that breaks on all of them. I have designed and printed new attachments but the installation of them can damage the motor if I'm not careful. The motor also looses torque after only a few months. The windings need to be improved.
Even though I really hate the plastic attachment, the button on top and the ability for it to stand up on its own without some stupid fiddly stand are the ONLY reasons I come back to this design. The moment someone makes a fully metal attachment (like I've seen) a power button on top and self standing, I'll throw this one away.
Updated April 2025: I am about to buy my 4th one since 2019. This is NOT an endorsement for it, I just haven't found anything design wise that's better. The motor in these are short lived and they give out with no warning. Ergonomically this design make more sense than a side mounted button or switch, but I'm still dropping a star for the fact I've had to buy 4 of these darn things in such a short time
Update: Oct 2025
I just bought this replacement in April and this things motor is already dead. I'm done with this item/seller. Just an overly cheap product. Product no longer worthy of even 2 stars
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Reviewed in the United States on May 27, 2024