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Description
Human NCF4 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 a Neutrophil cytosolic factor 4 (NCF4) 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 amount of Neutrophil cytosolic factor 4 (NCF4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Neutrophil cytosolic factor 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Neutrophil cytoplasmic factor 4 is a protein encoded by the NCF4 gene. This gene encodes a membrane-regulated component of the superoxide-producing phagocyte NADPH oxidase, a multicomponent enzyme system important for host defense. This protein is preferentially expressed in cells of the myeloid lineage. It primarily interacts with neutrophil cytoplasmic factor 2 (NCF2/p67-phox) and forms a complex with neutrophil cytoplasmic factor 1 (NCF1/p47-phox). The latter further interacts with the small G protein RAC1, translocating to the membrane upon cellular stimulation. This complex then activates aflatoxin b, the membrane-integrated catalytic core of the enzyme system. The PX domain of this protein can bind to the phospholipid product of PI kinases, suggesting a role in PI kinase-mediated signaling events. Phosphorylation of this protein has been found to negatively regulate enzyme activity. Alternatively spliced transcript variants encoding different isoforms have been observed. | |||||||||||||||||||||||||||||||||
| 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, and other biological fluids |
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4.4 ★★★★★
Based on 1696 reviews
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Product Reviews
★★★★★ 5
Durable Interactive Dog Toy with Bluetooth Sounds – Perfect for Dogs Who Love Cat-Style Toys
This interactive dog toy is surprisingly durable and very engaging, especially for dogs that love cat-style toys. My dog is obsessed with cat toys and also always wants to destroy something, so this toy is a perfect match. The Bluetooth sound feature makes it even more fun and interactive.
Pros
• Very durable main body made of hard plastic; my dog hasn’t been able to destroy it
• Great for dogs that like “mouser-style” toys
• Bluetooth feature lets you play animal sounds or music (I use Spotify dog playlists)
• Three different modes and very easy to switch
• Interactive and mentally stimulating
• Comes with two replaceable ropes, which I’m really happy about
Cons
• The ropes don’t last long for strong chewers
• The top part where the rope is attached can be chewed
Overall
Honestly, if there weren’t any parts my dog could destroy, he probably wouldn’t find the toy interesting. That’s why I’m actually happy it comes with two ropes that I can attach and replace. The ropes are inexpensive and easy to swap out, so it’s not really a downside for us. Overall, this is a fun and clever toy that keeps my dog entertained. 🐶
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Reviewed in the United States on January 5, 2026
★★★★★ 3
Great for 2 Days…see comments
So it’s great toy and had our dog entertained immediately. However probably won’t last very long with medium to large dogs that gets obsessed with killing every toy you give them. Our Pitt mix loved this until she ended it by chewing through the heavy plastic ring that keeps the ropes connected. Without the noted ropes attached, the toy cannot move around. Basically ending the fun. Would be great to start a puppy or smaller dog on. It has multiple modes and is noise and proximity activated. Our dog was obsessed but sadly it was no match for her. 3 stars only because it didn’t last more than two days with our dog.
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Reviewed in the United States on January 31, 2026
★★★★★ 2
Good but not tough
My dog loved it but he figured out how to open it pretty quickly. I think I’m doomed to never find an interactive dog toy that can survive my golden
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Reviewed in the United States on May 25, 2026
★★★★★ 4
Obsessed, crappy rope
My pitbull mix is obsessed! I was worried he might break it, but he has used it everyday for the past week and we've had no issues. The reason I docked a star is because the rope it comes with lasts minutes. I'm still trying to figure out a good solution of what to tie onto the top. Regardless, he loves it without anything on it, but he chews on the connector piece so I'm worried he will break it if I let him go.
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Reviewed in the United States on April 21, 2026
★★★★★ 5
Good For Heavy Chewers So Far
Color: Onyx Black - Most Durable
Solid quality. My Doberman is a power chewer and I am looking for a toy that he can't destroy in 2 seconds. If a ball lasts more than an hour it is a miracle. It has a sturdy design and is more heavy weight than the other "non-destructible" balls he has. He has only had his ball for one day so we will see how long this ball lasts him. It is the size of a tennis ball, firm, little to no bounce, and seems built for heavy chewing.
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Reviewed in the United States on January 30, 2026