Novus Biologicals' antibodies are the gold standard to monitor autophagy and detect LC3 expression. The recently published Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (3rd Edition) comprehensively details methods to monitor autophagy in cell or tissue samples. Importantly, these guidelines also provide key considerations for data interpretation and tips to creating better western blot data.
Use fresh samples and avoid multiple freeze thaws to improve detection of LC3-I when possible.
Why? LC3-I is less stable and more susceptible to freeze thaws and degradation in SDS compared to LC3-II. Moreover, most LC3 antibodies are less sensitive to LC3-I than LC3-II, impairing detection of LC3-I.
Use a PVDF membrane to improve detection of LC3-II.
Why? PVDF improves retention of LC3-II relative to nitrocellulose, possibly due to a higher affinity of PVDF for hydrophobic proteins such as LC3-II.
Normalize LC3-II expression to a loading control, not LC3-I.
Why? LC3-I expression levels differ from LC3-II between cell and tissue samples. In addition, LC3-I may be less sensitive than LC3-II to detection by some LC3 antibodies. These differences in detection and expression patterns make LC3-I an unsuitable candidate for normalization in most cases. We recommend actin as a loading control to normalize LC3-II expression.
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