The Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit provides an economical means of detecting target proteins related to autophagy vesicle elongation pathway. The kit contains enough antibody to perform two western blots per primary.
Specificity / Sensitivity
LC3A/B (D3U4C) XP® Rabbit mAb recognizes endogenous levels of total LC3A and LC3B proteins. Atg7 (D12B11) Rabbit mAb recognizes endogenous levels of total Atg7 protein. Atg4B (D1G2R) Rabbit mAb recognizes endogenous levels of total Atg4B protein. Atg4A (D62C10) Rabbit mAb recognizes endogenous levels of total Atg4A protein and recognizes unidentified bands within the molecular weight range of 48-60 kDa, which decrease with silencing of Atg4A expression. GABARAP (E1J4E) Rabbit mAb recognizes endogenous levels of total GABARAP protein, but does not cross-react with other GABARAP family members. Atg3 Antibody detects endogenous levels of total Atg3 protein.
Source / Purification
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu44 of human LC3B protein (conserved in LC3A), a synthetic peptide corresponding to residues near the amino terminus of human Atg7 protein, a synthetic peptide corresponding to residues surrounding Gln100 of human Atg4B protein, a synthetic peptide corresponding to residues near the carboxy terminus of human Atg4A protein, a synthetic peptide corresponding to residues surrounding Arg40 of human GABARAP protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of Atg3. Antibodies are purified by protein A and peptide affinity chromatography.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-8). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-9). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (10). Numerous mammalian counterparts to yeast Atg proteins have been described, including three Atg8 proteins (GATE-16, GABARAP, and LC3) and four Atg4 homologs (Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/autophagin-3, and Atg4D/autophagin-4) (10-12). The cysteine protease Atg4 is pivotal to autophagosome membrane generation and regulation (13). GABAA receptor associated protein (GABARAP) is an Atg8 family protein with a key role in autophagy, which was originally discovered as a protein associated with the GABAA receptor regulating receptor trafficking to the plasma membrane (14). Processing of GABARAP involves cleavage by Atg4 family members (15,16) followed by conjugation by the E1 and E2 like enzymes Atg7 and Atg3 (17,18).