Is sample dentaturation above 95°C, the golden standard?
The temperature of sample denaturation may influence the detected band pattern in Western blot. We recommend:
70°C for 5-10 minutes for total cell extracts from various plant tissues and algal cells. Higher denaturation temperatures may lead to protein aggregation, followed by lack of a specific band signal in Western blot.
Special care should be taken in case of membrane proteins, like H+ATPase, which may aggregate in high temperatures and thus never enter the gel.
Therefore, we recommend to carefully study instructions provided on the product information sheet for a given antibody.
Some antibodies may mostly work on proteins in native conformation and will therefore be suitable to use in techniques like ChIP and immunoprecipitation, while other antibodies will recognize mostly epitopes burried inside a protein and exposed by sample denaturation. Such details are important to pay attention to when chosing an antibody suitable for your experiments.