Product Pathways - Autophagy Signaling
Autophagosome Marker Antibody Sampler Kit #8666
|8666T||1 Kit (3 x 20 µl)||---||In Stock||---|
|Kit Includes||Quantity||Applications||Reactivity||Homology†||MW (kDa)||Isotype|
|Atg12 Antibody (Human Specific) #2010||20 µl||W, IF-IC||H||16, 55||Rabbit|
|LC3A (D50G8) XP® Rabbit mAb #4599||20 µl||W, IHC-P, IF-IC, F||H, M, R||Mk, Dg||14, 16||Rabbit IgG|
|LC3B (D11) XP® Rabbit mAb #3868||20 µl||W, IP, IHC-P, IF-IC, F||H, M, R||Mk, B, Pg||14, 16||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||W||All||Goat|
†Species predicted to react based on 100% sequence homology.
Applications Key: W=Western Blotting, IF-IC=Immunofluorescence (Immunocytochemistry), IHC-P=Immunohistochemistry (Paraffin), F=Flow Cytometry, IP=Immunoprecipitation
Reactivity Key: H=Human, M=Mouse, R=Rat, All=All Species Expected
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from various cell lines using Atg12 Antibody (Human Specific).
Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or chloroquine-treated (50 μM, overnight), using LC3A (D50G8) XP® Rabbit mAb.
Western blot analysis of extracts from various cell lines, untreated (-) or treated overnight with chloroquine (50 μM) (+), using LC3B (D11) XP® Rabbit mAb (upper) or LC3B Antibody #2775 (lower).
Confocal immunofluorescent analysis of HCT-116 cells, untreated (left) or chloroquine-treated (right), using Atg12 Antibody (Human Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded human glioblastoma multiforme using LC3A (D50G8) XP® Rabbit mAb.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® LC3B siRNA I #6212 (+) or SignalSilence® LC3B siRNA II #6213 (+), using LC3B (D11) XP® Rabbit mAb #3868 and α-Tubulin (11H10) Rabbit mAb #2125. The LC3B (D11) XP® Rabbit mAb confirms silencing of LC3B expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of LC3B siRNA.
Immunohistochemical analysis of paraffin-embedded human astrocytoma using LC3B (D11) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using LC3A (D50G8) XP® Rabbit mAb (left) or
Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or chloroquine-treated (right), using LC3B (D11) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or choloroquine-treated (right), using LC3A (D50G8) XP® Rabbit mAb.
Flow cytometric analysis of HeLa cells using LC3B (D11) XP® Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
The Autophagosome Marker Antibody Sampler Kit provides an economical means to investigate the accumulation of autophagosomes within the cell. The kit contains enough primary and secondary antibodies to perform four western blots per primary antibody.
Specificity / Sensitivity
Each antibody in the Autophagosome Marker Antibody Sampler Kit detects endogenous levels of total respective protein. Atg12 Antibody (Human Specific) recognizes free Atg12 and the Atg12-Atg5 conjugate. LC3A (D50G8) XP® Rabbit mAb and LC3B (D11) XP® Rabbit mAb may cross-react with other LC3 isoforms and have stronger reactivity with the type II form.
Source / Purification
Antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Atg12 protein, residues near the amino terminus of human LC3A protein, or residues near the amino terminus of human LC3B protein. Polyclonal 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 has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). The molecular machinery of autophagy was largely discovered in yeast and referred to as autophagy-related (Atg) genes. Formation of the autophagosome involves a ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagosome vesicles (4-6). This conjugation reaction is mediated by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10 (7,8). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (9) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 that is critical for autophagy (10). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (11-14). 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 (11-15). 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 (16).
- Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
- Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
- Levine, B. and Yuan, J. (2005) J Clin Invest 115, 2679-88.
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- Suzuki, K. et al. (2001) EMBO J 20, 5971-81.
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- Mann, S.S. and Hammarback, J.A. (1994) J Biol Chem 269, 11492-7.
- Lang, T. et al. (1998) EMBO J 17, 3597-607.
- Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
- Wu, J. et al. (2006) Biochem Biophys Res Commun 339, 437-42.
- He, H. et al. (2003) J Biol Chem 278, 29278-87.
- Tanida, I. et al. (2004) J Biol Chem 279, 47704-10.
- Ichimura, Y. et al. (2000) Nature 408, 488-92.
- Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
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