Western Blot Protocol Page for Protein Detection
Western blotting, also known as immunoblotting, is a widely used technique for detecting and quantifying specific proteins in a complex mixture and validating antibodies. This protocol outlines the key steps involved in performing a Western blot and serves as a guide for researchers and customers of antibody products.
*Make sure to use personal protective equipment, such as gloves and lab coats.
1. Perform SDS-PAGE to separate proteins based on their molecular weight. After electrophoresis, remove the gel from the cassette.
2. Prepare the transfer apparatus. Place the gel and membrane in the transfer sandwich with the transfer buffer. Apply a constant voltage to transfer proteins from the gel to the membrane (typically 100 V for 1–2 hours).
3. Incubate the membrane in blocking solution to prevent non-specific binding. Block for 1 hour at room temperature or overnight at 4 °C.
4. Incubate the membrane with the specific primary antibody. Dilute the primary antibody according to the manufacturer’s recommendations. Incubate for 1–2 hours at room temperature or overnight at 4 °C with gentle agitation.
5. Wash the membrane with a suitable buffer to remove unbound primary antibody.
6. Incubate the membrane with the appropriate secondary antibody. Dilute the secondary antibody according to the manufacturer’s recommendations. Incubate for 1 hour at room temperature with gentle agitation.
7. Wash the membrane to remove unbound secondary antibody.
8. Detect the protein bands using the chosen detection method (e.g., chemiluminescence or fluorescence). Capture images or obtain data as needed.
Analyze the Western blot data to determine the presence and quantity of the target proteins. However, it is important to point out that validating an antibody for Western blotting is crucial to ensure accurate and reliable results. Here are four key steps to validate an antibody:
By following these steps, you can thoroughly validate an antibody for Western blotting, increasing the reliability of your experimental results.