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Description
Human NF-kB 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: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled 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 freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freezing and thawing. 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 enzyme conjugate are sequentially added to microwells pre-coated with a Nuclear Factor Kappa B (NF-kB) 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 Nuclear Factor Kappa B (NF-kB) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Nuclear Factor Kappa B ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | NF-κB (nuclear factor kappa B) is a protein complex that controls DNA transcription, cytokine production, and cell survival. It is present in nearly all cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, UV exposure, oxidized low-density lipoproteins, and bacterial or viral antigens. NF-κB plays a key role in regulating the immune response to infection, and its misregulation has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infections, and inappropriate immune development. NF-κB is also involved in synaptic plasticity and memory processes. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.4 ★★★★★
Based on 28 reviews
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Product Reviews
★★★★★ 5
Sturdy Quality
Style: Cake Shape, Style: Cake Shape
These toys were perfect for my dog’s 4th birthday. You get three toys for a really reasonable price, and the quality is actually really good. He loved them right away, especially the cake toy — it makes a great crinkle sound that he can’t get enough of. They were also perfect for birthday photos. Overall, a really cute set and definitely worth it.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 23, 2026
★★★★★ 5
Loved By Maizy
Style: Monkey Shape
Pup loved this monkey! Did not take her long to get the stuffing and squeaker out but she still loves what is left of it.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 12, 2026
★★★★★ 5
If your dog pops with his canines and shreds with his front teeth, this is for you. I have nothing but praise for this toy.
Size: One Size, Style: Bumpy Ball
This is one of the few balls I have purchased that my dog does not destroy. I am surprised at the negative reviews here and have been doing some thinking about why one dog destroys this ball and another does not. Perhaps it is the type of chewer we own? Or because he has a narrow jaw rather than a broad one like a pit bull? My Airedale Terrier likes to shred, preferring to use his back teeth to pop or crush and front teeth to pull apart tennis balls, stuffed animals and the like. I don't know if that will help anyone in making the decision to buy or not to buy this toy. The ball has many dimples where my dog's teeth have sunk in but he has yet to take a chunk out of it. I bought two of these from Marshall's three or four months ago and I play with my dog twice a day, so lots of use here. After I realized what I'd found I promptly ordered two more from Amazon because I wanted to insure I would have them around for a long time. All I can say is that this toy has been a lifesaver for me.
My extremely active one year old Airedale Terrier needs a lot of exercise and this ball satisfied that need for us. It is fun for him and I giggle at his antics as he attempts to follow the unpredictable path of the bumble ball. I throw the ball for him from my porch, trying to toss it so it goes over our truck, which my husband leaves in the driveway. If the ball hits the truck, no harm done and more unpredictable for my boy! I can toss the ball around the house without worrying it will break a window. My dog loves to pick up toys and throw them in the air when he plays. He tossed a marrow bone into the air and when it landed I had a nice half inch gash in our coffee table. No more marrow bones in the house for him.
I have nothing but praise for this toy and am grateful to have found it.
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Reviewed in the United States on May 3, 2016
★★★★★ 5
Great Toy
Size: One Size, Style: Stick
I breed German shepherds. This toy is a soft formed stick made of a memory foam/ rubber polymer. It’s light weight, very durable, floats and my German Shepherds love retrieving this toy. They are not indestructible but they are a great chewing toy for their price. I usually buy several at a time. This toy will help clean their teeth and is soft enough not to damage their teeth. It’s a favorite item for my dogs.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 9, 2024
★★★★★ 5
SPECTACULAR dog chew toy!
Size: One Size, Style: Ball
If you look at my profile, you will see that I have bought and reviewed a LOT of dog toys! My boxer is an "aggressive" chewer and we go through dog toys quickly in my house. This has been one of my very favorite chew toys that I have ever purchased. This company sells quite a few of these chew toys in different shapes. This ball is made from a very light foam rubber (I think) material. When I showed it to my husband, he said "that won't last 5 minutes" and I had my doubts too. However, this ball has proven to be EXTREMELY durable! Because it is made from the foam material, it has a lot of tooth sized holes in it after she chews it, but she has been unable to chew the toy apart. I bought another of these chew toys made from the same material in a different shape thinking surely there was some accident that the toy had held up for so long. Nope, the new toy has held up just as well! The toys float in water and are very light and easy to throw. Overall, I will be buying more of these toys and would recommend this product!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 27, 2015
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