Cell Signaling Technology

Product Pathways - Autophagy Signaling

Autophagosome Marker Antibody Sampler Kit #8666

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Atg12 Antibody (Human Specific) #2010 40 µl W IP IF-IC H 16, 55 Rabbit
LC3A (D50G8) XP® Rabbit mAb #4599 40 µl W IHC-P IF-IC F H M R (Mk) (Dg) 14, 16 Rabbit IgG
LC3B (D11) XP® Rabbit mAb #3868 40 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 Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  Dg=Dog  Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg12 Antibody (Human Specific) #2010.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines, untreated (-) or treated with chloroquine (50 μM, overnight; +), using LC3B (D11) XP® Rabbit mAb #3868.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3A (D50G8) XP® Rabbit mAb #4599.


Description

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.

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.

Background

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).

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  3. Levine, B. and Yuan, J. (2005) J Clin Invest 115, 2679-88.
  4. Mizushima, N. et al. (1998) J Biol Chem 273, 33889-92.
  5. Mizushima, N. et al. (1998) Nature 395, 395-8.
  6. Suzuki, K. et al. (2001) EMBO J 20, 5971-81.
  7. Tanida, I. et al. (1999) Mol Biol Cell 10, 1367-79.
  8. Shintani, T. et al. (1999) EMBO J 18, 5234-41.
  9. Mann, S.S. and Hammarback, J.A. (1994) J Biol Chem 269, 11492-7.
  10. Lang, T. et al. (1998) EMBO J 17, 3597-607.
  11. Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
  12. Wu, J. et al. (2006) Biochem Biophys Res Commun 339, 437-42.
  13. He, H. et al. (2003) J Biol Chem 278, 29278-87.
  14. Tanida, I. et al. (2004) J Biol Chem 279, 47704-10.
  15. Ichimura, Y. et al. (2000) Nature 408, 488-92.
  16. Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.

Application References

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Protocols

* Product-specific protocol.

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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