Breakdown: Interpreting LC3 Antibody WB Results

In rodents, MAP1LC3 (Microtubule-associated protein 1 light chain 3) is expressed in the renal visceral epithelial cells, or podocytes. LC3 antibody analysis has shown the protein accumulates in its membrane-bound form, LC3II, following conversion from LC3I. LC3II antibodies, therefore, play an important role in autophagy research as useful markers for autophagic activity.

Autophagy is the bulk degradation, or breakdown, of proteins and organelles and is an essential part of normal cellular maintenance, aiding cell growth, differentiation and viability. It, however, is also a feature of many human clinical conditions, including neurodegenerative and neuromuscular disease, cardiomyopathies, cancer, and viral and bacterial infections. A key part of the process is the formation of an autophagosome, which is controlled by two ubiquitination-like processes – conjugation of ATG12 and conversion of LC3. LC3 is an orthologue of yeast ATG8, the other orthologues being GABARAP and GATE-16.

LC3 antibody immunoblot assays, which measure conversion of LC3-I into LC3-II, are routinely used in research on tumorigenesis. The amount of LC3II detected is considered to correlate with the number of autophagosomes being formed; however, LC3 antibody research has shown that LC3-II is itself degraded during the autophagy process, making interpretation of results difficult. Also, the presence of LC3 is not necessarily an indication of autophagic flow; therefore LC3-II levels should be measured in the presence and absence of lysosomal protease inhibitors.

In addition, immunoblot LC3 antibody detection is more sensitive to LC3-II than LC3-I, further skewing the results. When purchasing LC3 antibodies from Novus Biologicals, consider alternative assays, such as Immunofluorescence.

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