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Description
Rat 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, 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 200 pg/mL). Then dilute to the following concentrations: 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL, 6.25 pg/mL, 3.125 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 200pg/mL standard working solution into the first EP tube and mix thoroughly to make a 100pg/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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| Sensitivity | 1.75 pg/mL | |||||||||||||||||||||||||||||||||
| 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 (TNF-a) 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 (TNF-a) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Tumor Necrosis Factor Alpha ELISA Kit，Rat TNF-a 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. The naturally occurring form of TNF-α is glycosylated, but nonglycosylated 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 exhibit 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 | 3.12-200 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.6 ★★★★★
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Product Reviews
★★★★★ 5
Held up for well for 5 months. Fit my son’s chubby feet like no other shoe.
My son has been wearing this shoe since he was 9 months old and was cruising furniture. I bought the 6-12 month size. He started walking at 10 months and is 14 months now. He’s still wearing this shoe and it has held up well. I have him barefoot in the house and mostly use it for when he’s in the back yard, at the gym childcare and when we are out and about. He wears shoes for several hours a day at least, though I bought the Robeez sandals at the start of summer and have been switching between the two pairs. My son has wide feet and doesn’t really fit in anything but soft sole shoes. His feet are about 5” long now and these shoes are getting too small. It’s almost time to size up! They’ve treated us well. Plus they are super cute and match every outfit. He giggles when I put them on.
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Reviewed in the United States on August 1, 2019
★★★★★ 5
Cute, different, and good quality
I had these for my kids when they were little. I am now buying them for my first grandchild. They appear to be the same good quality they were years ago. I highly recommend : )
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Reviewed in the United States on August 21, 2025
★★★★★ 5
Great shoes for the little one taking his/her first steps
Great shoes for the little one taking his/her first steps!! I actually ordered 2 pairs, one for my son in the 12-18 months (even tho he was 10 months old) and a 6-12 month for a friend. The 6-12 month fit my son perfect!! (my son was in a size 5) but I knew they would only last a few weeks so I kept the 12-18 month ones and he started wearing them. He seemed to like them, never tried taking them off and they were easier to walk in than socks but for some reason the top of the foot area is HUGE! They stayed on no problem but lots of wiggle room in the top... He is now running and wears shoes with a thicker sole for the rocks but we still love these for inside when it is too cold to go barefoot. I would say the 12-18 month fit like an average size 6.
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Reviewed in the United States on May 11, 2017
★★★★★ 5
Great barefoot option for early walkers!
Size: 12-18 Months Infant, Color: Grey
A moccasin is such a great place to start! Buying barefoot shoes can be hard.. they’re not always the cutest, can be expensive & there’s a lot to pay attention to! We love moccasins as a great first shoe for our boys. Perfect for summer because they’re more like a sandal & let in air flow.
Why we love:
✨Meets barefoot-style checklist
✨SUPER inexpensive
✨Quality made & last a long time
✨Lots of style & sizing options
✨Breathable
✨Easy to put on & take off
✨They protect feet all while still giving a sensory experience
These ones do run big, so size down. If you’re looking for a shoe that has more grip on the bottom, there are tons of moccasin style shoes with grip as well.
Give these a shot if you have a fresh walker on your hands!
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Reviewed in the United States on June 5, 2025
★★★★★ 5
Great shoes for first time walkers!
Size: 12-18 Months Infant, Color: Grey, Size: 12-18 Months Infant, Color: Grey
The material is great! My child likes it and hasn’t tried to take them off! They are lightweight and not bulky. Very flexible ! Also I did my research and it’s made in the UK and made with genuine leather! The color is cute as well! Wish they did have more variety here in Amazon but still okay with it!
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Reviewed in the United States on April 30, 2026