LC3 is a mammalian homolog of the yeast protein Atg8, first identified as a light chain of the microtubule-associated proteins MAP1A/B. The only known marker for both the inner and outer autophagosome membrane, at least three mammalian transcript variants are known to exist. Antibody suppliers now offer a wide choice of LC3 antibodies for the identification and tracking of autophagic activity.
One of the most commonly used ways to monitor mammalian autophagy is to track the conversion of LC3 by immunoblotting, e.g Western blot analysis. However, LC3 itself is not normally detectable as it undergoes Atg4 modification immediately after synthesis. In fact, SDS-PAGE and immunoblot analysis reveal two distinct bands: LC3-I (16 – 18 kDa) and LC3-II (14 – 16 kDa). These offer a convenient autophagy tracking system.
Following synthesis, LC3 is cleaved at the carboxy terminus, yielding the cytosolic LC3-I form. During autophagy, LC3-I is converted to the active form of LC3-II, associating with the autophagosomal membranes. This is achieved by a ubiquitin-like process, during which LC3-I undergoes covalent linkage to the phosphatidylethanolamine (PE) lipid. Therefore when monitoring LC3 conversion, it is PE-conjugation, rather than processing of LC3, which is being measured. Recent antibody research suggests WIP12, the human homolog of Atg18, is necessary for this, positively mediating LC3 lipidation.
LC3-II has a higher molecular weight than LC3-I, but in SDS-PAGE shows a faster migration rate, probably due to its extreme hydrophobic qualities. Therefore LC3 antibodies offer a reliable method to both detect autophagy and track its progression. LC3-II antibody levels are a particularly good indicator of autophagosome formation, owing to the short half-life of LC3, its close association with the autophagosomal membrane, and the transient nature of the autophagosome itself. However, caution must be exercised when comparing LC-I and LC3-II levels, as LC3-II is itself degraded during autophagy. Also, LC-I and LC3-II differ in antibody sensitivity during immunoblotting.
We at Novus Biologicals have an extensive antibody catalog, and are continually expanding our LC3 antibody database to take account of the latest autophagy research.
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