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Description
Mouse TNF-α ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000xg for 20 minutes, and remove the supernatant for testing. (10ul sample load: Serum: Place the whole blood sample collected in the serum separator tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes. Remove the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant, and centrifuge at 1000×g for 15 minutes at 2-8℃ within 30 minutes of collection. Remove the supernatant for testing, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing.) Preparation before testing: 1. Please remove the test kit from the refrigerator 10 minutes in advance and equilibrate it to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 1000 pg/mL). Then dilute to the following concentrations: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 1000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500 pg/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 Tumor Necrosis Factor Alpha (TNFa) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Tumor Necrosis Factor Alpha (TNFa) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Tumor Necrosis Factor Alpha ELISA Kit，Mouse TNFa ELISA Kit，TNF-a | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Tumor necrosis factor alpha (TNF-α) is produced by neutrophils, activated lymphocytes, macrophages, natural killer (NK) cells, lymphocyte kinase (LAK) cells, astrocytes, endothelial cells, smooth muscle cells, and some transformed cells. Mouse TNF-α is present in a membrane-anchored form. The naturally occurring form of TNF-α is glycosylated, but non-glycosylated recombinant TNF-α exhibits comparable biological activity. The biologically active native form of TNF-α is reported to be a trimer. Human and murine TNF-α display approximately 79% homology at the amino acid level, and cross-reactivity is observed between the two species. | |||||||||||||||||||||||||||||||||
| 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 | 15.6-1000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids (10ul loading serum, plasma) |
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4.7 ★★★★★
Based on 1448 reviews
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Product Reviews
★★★★★ 5
Breathe Easy!
Style: BE-728H 1-Pack
Ladies and gentlemen, buckle up, because we're about to embark on a whirlwind adventure of cleaner air and contortionist-worthy installation feats with the Spearhead HEPA Breathe Easy Cabin Filter!
First off, let's talk about installation. If you're someone who loves a good yoga session or considers themselves a Cirque du Soleil dropout, this is the filter for you. The instructions are so clear and helpful that even a goldfish could follow them. However, be prepared to twist and turn in ways you didn't know your body could. And let me tell you, I'm not in the best of shape. I discovered muscles I never knew I had and positions that would make a pretzel jealous. But don't worry, it's all worth it.
Once installed, my car’s air quality shot up faster than a rocket to Mars. And although my car is old and doesn't have any fancy AI, it felt like it was saying, "Ahh, finally, some quality air!" The air was so crisp and pure, I half expected to see little cartoon birds chirping around my head. And let me emphasize this: the product quality is top-notch. It not only works great but looks good too.
I purchased this gem because my wife has bad allergies, and so far, it's been a game-changer. She hasn't sneezed once, and the car doesn't smell as musty with the air conditioning on. It's like we've been transported to a fresh, alpine air paradise every time we drive.
In summary, the Spearhead HEPA Breathe Easy Cabin Filter is a game-changer. It's as easy to install as putting on socks—if putting on socks required you to become a human origami. But trust me, once it's in place, your lungs will thank you, and your car will feel like it's breathing the fresh, alpine air of the Swiss Alps. Five stars, and a standing ovation from my now incredibly limber body!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on July 10, 2024
★★★★★ 5
Works a lot better than the previous one.
Style: BE-775H 1-Pack
Great product at a great price. It seemed to fit almost perfect with out having to crunch it in. Highly recommend.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 5, 2026
★★★★★ 5
Great Cabin Filter Upgrade
Style: BE-729H 1-Pack, Style: BE-729H 1-Pack
I recently replaced my old cabin air filter with this one, and the difference was noticeable right away. Installation was quick and straightforward took less than 10 minutes with no special tools needed.
What I appreciate most is the air quality improvement. The airflow feels stronger, and there’s a clear reduction in dust, odors, and outside pollution entering the cabin.
If you’re looking for an easy, affordable upgrade that actually makes a difference in everyday driving comfort, this cabin filter is definitely worth it.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 19, 2025
★★★★★ 5
Excellent Air filter - Easy installation.
Style: BE-743 1-Pack
Excellent AC and Heater Cabinet odor defense air filter. I noticed immediately the smell coming out of the vents was fresher and the musty smell that was coming from the vents was crisper and cleaner. Very easy installation that you can do yourself and don’t need to pay a shop to do. The price is one of the best online and coms in quickly. It’s very sturdy, filter quality is excellent and it definitely improves the air quality and flow in your car. Fits perfectly like any OEM filter, this is a great buy for the price of an installation that will take you Les then 10 minutes to do yourself.
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Reviewed in the United States on May 21, 2026
★★★★★ 5
Great filter and easy installation
Style: BE-159 1-Pack, Style: BE-159 1-Pack
For my 2016 Hyundai Sonata Hybrid Limited. I cannot believe I paid so much more for a brand-name filter that does the same thing as this one. It is made of the same material as the dealer one I took out. Very easy to install, cant believe I paid for this to be done in the past with oil changes. Requires no tools and did not get dirty at all. No mechanical skills needed, especially if you run into trouble and can use YouTube.
PROS
- great quality, cannot tell that it is after-market
- very professionally made, not irregularities seen
- excellent fit in position, no adjustment needed
- very easy to install, cannot believe I paid to have this done in the past
- no mechanical skills needed to install
- great price compared to what big name productions want for the same thing
- cannot tell the difference in air quality between this and the dealer filter I took out
- removes outside odor completely
- did not require tools are getting dirty
CONS
- None
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 30, 2026
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