1.3 Protein Denaturation

Protein Denaturation
Experimental Procedures
1. Denaturation of Western Blot (WB) Samples
If a denaturing gel is used for WB, the samples must be denatured before loading. The denaturation method involves adding a protein loading buffer containing denaturants such as β-mercaptoethanol (β-ME) and dithiothreitol (DTT), followed by boiling at 95–100°C for 5 minutes. For samples containing multi-transmembrane proteins, heating at 70°C for 10 minutes is recommended instead.
2. Denaturation of Samples for WB After Immunoprecipitation (IP)
Add a protein loading buffer mixed with denaturants like β-ME and DTT to the samples after IP. Heat the samples to 95–100°C and maintain this temperature for 2–5 minutes to ensure complete denaturation. Since the samples contain relatively large beads, cut off the tip of the pipette with scissors before aspirating the samples. Before loading, pipette the samples up and down to mix them evenly.

Note: This step can be omitted if non-denaturing gel electrophoresis is performed.