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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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4.5 ★★★★★
Based on 30 reviews
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Product Reviews
★★★★★ 5
Real steel pipe
Size: O Desk Unfinished 3/4-in and 1/2-in
This pipe table leg set was great. It's real steel pipe like from the hardware store. It does have a layer of oil on it so that must be stripped using dawn or some other degreaser before handleing and painting. It is also offset in the middle bar so if you use it as a desk you get more leg clearance. I used it as a table base so I bought matching middle braces to set the middle support in the center of the table. Once together it looks great. I highly recommend getting this if you want a good quality pipe base.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 11, 2023
★★★★★ 5
Price / Quality - Office Desk - Perfect
Size: H Desk Unfinished 3/4-in and 1/2-in
Purchased two sets to create a custom L shaped desk for the office. Placed a pine stained top. Perfect to handle a monitor, multiple laptops, normal office equipment. Extra benefit is the cross piece at the bottom as a foot rest. Very sturdy while sitting on carpet. Compared to normal pipe material purchased in large box store -- could not have asked for a better product for the price. Looking at other products for additional projects for around the house for the product and vendor. - Highly Recommend.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 27, 2021
★★★★★ 4
Fantastic style, good stability, customizable finish, and great customer service
Size: M Desk Unfinished 3/4-in and 1/2-in
Put this together in about April 2020 right about the crazy lockdown/quarantine/work-from-home timeframe. I knew that I wanted a simple hardwood desk with no drawers or stuff below the top. I have an ergonomic keyboard tray that I have attached to all my work desks for many years now, and it was too long for my old home desk, which was also made of MDF and was falling apart. Boo.
Enter this kit plus a butcher block top from the big box store. This kit went together easily. I had lost the instructions but a quick email to the JS Supplies company, and they hooked me up. By the time they sent it, I had actually figured out the part that was puzzling, which was what the all-thread pieces were for (the couplers between the elbows and the flanges on the top side).
I degreased all the parts with a simple Simple Green-type solution diluted in a 5 gallon bucket. I then sprayed the pieces with a satin nitrocellulose laqcuer (Minwax brand that I got from the home improvement big box). Worked great. I thought about doing a sealed rust finish using a science project volcano--vinegar and baking soda for a quick layer of rust on the outside--but I decided against it, as I wasn't sure of how stable the final product would be, even after a clear spray finish. The satin nitro finish looks great without even looking like a finish. I also coated the pieces with paste wax after the finish dried, just for good measure.
The one thing that is a bit disappointing is the stability... WITH MY TOP, mind you.I should be clear that the top that I'm using is a 1-3/4" thick monster piece of birch butcher block that is 60" x 30". I'm guessing that it's 40-50 pounds? Maybe more. This is probably WAY more weight than this leg set is made for. I also have the aforementioned keyboard tray that sticks out (torque equals force times lever arm) and causes even more torque. I get some shaking with the top with this setup. I have the desk on carpet; I was concerned that maybe the carpet and pad were not giving enough stability with the feet, so I made some plywood platforms for the feet. I also carefully leveled the feet to make sure it wasn't just uneven legs that was causing the instability. Still shakes no matter what I tried.
I think it's just that I have such a heavy top--more than this thing is spec'd for. That's totally fine, and I don't want to knock off a bunch of stars for that. I leave these comments more as a "don't do what I did" thing. Just be advised that you need to limit the weight of the top to something reasonable.
The design of one upright on each side of the desk with a pipe coupler in between is probably the weak spot with my heavy desktop; I'm thinking of either adding tension on either side (with some threaded rod and turnbuckles) or some extra supports with something like the Steel-Tek pieces from Lowe's.
Overall, I'm very satisfied with the product and the company!
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Reviewed in the United States on June 23, 2020
★★★★★ 5
So nice I bought twice!
Size: H Desk Unfinished 3/4-in and 1/2-in, Size: H Desk Unfinished 3/4-in and 1/2-in
For starters, this is a great buy if it is exactly what you want. For me that was the case and it would have cost me a good $40 or more to replicate it at a big box store. With that said, if you are not planning on using all of the pieces and want a different configuration, this may not be the kit for you. All of the parts inside are just like if you bought it at a local home improvement store. All the pipes need to be cleaned and protected. I personally found that dish soap (dry off immediately after you clean a piece) was the easiest to clean everything and I finished it with some wax. The kit is sturdy and easily supports the weight of my butcher block tops. I inverted the kit as it is designed as I intend on adding a shelf and am planning on using the support brace to aid in holding that shelf up for cable management. After cleaning and protecting, putting the kit together only took 5-10 minutes and that was with kid disruptions. I purchased 2 so that my wife and I could have matching desks in the office and I couldn't be happier.
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Reviewed in the United States on August 23, 2019
★★★★★ 5
Durable and sturdy. I bought two
Size: H Desk Unfinished 3/4-in and 1/2-in
Love the kit. I ordered two to make an L-shaped desk for my home office. Perfect height, looks cool, and sturdy. Slightly tricky to get two of them to the exact same height but you can adjust them buy loosing/tightning parts. Way cheaper than going to the hardware store and buying the same parts. I wiped each piece down as they are a bit dirty/greasy, but no big deal.
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Reviewed in the United States on August 9, 2021
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