The Mouse TNF Alpha ELISA Kit is a powerful tool for quantifying TNF-alpha, a key inflammatory cytokine, in mouse samples. Accurate TNF-alpha measurement is essential for research in immunology, inflammation, and disease models. To achieve reliable results, follow this detailed, step-by-step protocol.
Materials Needed
- Mouse TNF Alpha ELISA Kit (includes pre-coated 96-well plate, standard, detection antibody, enzyme substrate)
- Mouse serum, plasma, or cell culture supernatant samples
- Wash buffer (included or prepared as specified by the kit instructions)
- Plate reader (capable of reading absorbance at 450 nm)
- Pipettes and tips (adjustable for precision)
- Plate shaker (optional for better mixing)
Step-by-Step Protocol
1. Preparation of Reagents and Samples
- Reconstitute Standards: Dilute the TNF-alpha standard as per the kit’s instructions to create a standard curve with serial dilutions. This usually ranges from high to low concentrations.
- Prepare Samples: If needed, dilute your samples with the provided buffer. Use the suggested dilution factor based on expected TNF-alpha concentration.
- Prepare Detection Antibody and Enzyme Conjugate: Dilute the detection antibody and enzyme conjugate as directed. Only prepare the amount needed to minimize waste and preserve stability.
2. Plate Preparation
- Each well of the 96-well plate is pre-coated with anti-mouse TNF-alpha capture antibody.
- Plan the layout of standards, samples, and controls. Run each in duplicate or triplicate for accuracy.
3. Adding Standards and Samples
- Add Standards: Pipette 100 µL of each TNF-alpha standard into the designated wells.
- Add Samples: Pipette 100 µL of each prepared sample into its designated well.
- Add Controls: Include blank and negative controls in the plate as recommended by the protocol.
4. Incubation
- First Incubation: Cover the plate with a plate sealer and incubate at room temperature or at the temperature specified in the protocol. This incubation allows the TNF-alpha in the samples to bind to the capture antibody on the plate.
- Optional Shaking: Placing the plate on a shaker can improve mixing and binding efficiency.
5. Washing
- After incubation, wash the plate 3-5 times with the wash buffer to remove unbound material.
- Use an automated plate washer if available, or manually wash by adding wash buffer, shaking out contents, and blotting on absorbent paper. Proper washing is crucial to reduce background noise.
6. Adding Detection Antibody
- Add 100 µL of the diluted detection antibody to each well.
- Second Incubation: Incubate the plate again at room temperature for the specified time to allow the detection antibody to bind to TNF-alpha in the wells.
- Wash: Repeat the washing steps to remove excess detection antibody.
7. Adding Enzyme Conjugate
- Add 100 µL of the enzyme conjugate solution to each well.
- Third Incubation: Incubate at room temperature for the specified time to allow binding.
- Wash: Repeat the wash steps thoroughly to remove any unbound enzyme conjugate.
8. Substrate Addition
- Add 100 µL of the enzyme substrate solution to each well. The substrate will react with the enzyme conjugate to produce a color.
- Final Incubation: Incubate the plate in the dark, avoiding light exposure, as per the protocol’s recommended time.
9. Stopping the Reaction
- Add 50 µL of stop solution to each well. This stops the color development and stabilizes the color for reading.
- Note: The stop solution will change the color from blue to yellow, enhancing the readout at 450 nm.
10. Plate Reading and Analysis
- Measure Absorbance: Using a plate reader, measure the absorbance at 450 nm (with a reference wavelength at 540 or 570 nm if specified).
- Create Standard Curve: Plot the absorbance values of the TNF-alpha standards to create a standard curve. Use this curve to determine the concentration of TNF-alpha in your samples.
- Analyze Data: Calculate sample concentrations based on the curve, adjusting for any dilution factors used in sample preparation.
Tips for Reliable Results
- Accurate Pipetting: Ensure all volumes are precise to maintain consistency.
- Consistent Timing: Incubate each plate for the exact times specified to avoid variation.
- Proper Washing: Inadequate washing can increase background noise, while excessive washing may reduce signal.
- Run in Duplicates/Triplicates: Redundant samples increase reliability by reducing the effects of random errors.
By following these steps and tips, the Mouse TNF Alpha ELISA Kit can provide consistent, reliable results, helping you accurately quantify TNF-alpha levels and gain valuable insights in your research.
