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Western Blot of non-enriched lysate and LC3-II-enriched protein fraction from HeLa cells prepared with the LC3-II Enrichment Kit (Western Blot) (Catalogue No. 17-10232). Proteins were subjected to SDS-PAGE and were transferred to a PVDF membrane. The membrane was probed with primary anti-LC3 and anti-TOMM22, followed by secondary antibodies. The blot was developed by enhanced chemiluminescence. Immunoblotting results of non-enriched lysates indicate the LC3-I signal decreases over time after induced autophagy, as the LC3-II signal increases. After enrichment, the LC3-I signal is no longer detectable and the LC3-II signal is retained. (Click image to enlarge.)
LC3-II Enrichment Kit (Western Blot)
(Catalogue No. 17-10232)
LC3 precursors are proteolytically processed to form LC3-I, which is diffusely distributed in the cytosol. Upon initiation of autophagy, the C-terminal glycine of LC3-I is modified by addition of a phosphatidylethanolamine (PE) to form LC3-II, which translocates rapidly to nascent autophagosomes in a punctate distribution.
However, detecting and interpreting the relative amounts of LC3-I and LC3-II in standard assay methods can be complicated. When LC3 is immunochemically detected by Western Blotting following SDS-polyacrylamide gel electrophoresis, LC3-II typically appears as a slightly lower band than the 18 kDa LC3-I band as a result of the greater hydrophobicity of LC3-II.
However, in some instances, the LC3-I and LC3-II bands are incompletely resolved, which complicates analysis. Also, even when LC3-I and LC3-II are adequately separated on an immunoblot, changes in the relative ratios of the isoforms may not be directly proportional to autophagosome quantity, due to differential immunoreactivity of the isoforms. MilliporeSigma’s LC3-II Enrichment Kit (Western Blot) enables sensitive and accurate quantification of autophagosome density by utilizing a selective permeabilization procedure that removes cytosolic LC3-I and retains autophagosome-bound LC3-II. This procedure reveals quantities of LC3-II, without interference from LC3-I, by Western blotting analysis.
However, in some instances, the LC3-I and LC3-II bands are incompletely resolved, which complicates analysis. Also, even when LC3-I and LC3-II are adequately separated on an immunoblot, changes in the relative ratios of the isoforms may not be directly proportional to autophagosome quantity, due to differential immunoreactivity of the isoforms.
MilliporeSigma’s LC3-II Enrichment Kit (Western Blot) enables sensitive and accurate quantification of autophagosome density by utilizing a selective permeabilization procedure that removes cytosolic LC3-I and retains autophagosome-bound LC3-II. This procedure reveals quantities of LC3-II, without interference from LC3-I, by Western blotting analysis.
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