| Product Includes | Product # | Quantity | Color |
|---|---|---|---|
| FastScan™ ELISA Microwell Strip Plate, 96 Well | 53257 | 96 tests | |
| LC3B Rabbit Capture mAb | 48369 | 1 ea | Green (Lyophilized) |
| LC3B Mouse HRP-linked mAb | 59959 | 1 ea | Red (Lyophilized) |
| FastScan™ ELISA Capture Antibody Diluent | 16076 | 3 ml | Green |
| FastScan™ ELISA HRP Antibody Diluent | 28120 | 3 ml | |
| TMB Substrate | 7004 | 11 ml | |
| STOP Solution | 7002 | 11 ml | |
| ELISA Wash Buffer (20X) | 9801 | 25 ml | |
| FastScan™ ELISA Cell Extraction Buffer (5X) | 69905 | 10 ml | |
| FastScan™ ELISA Cell Extraction Enhancer Solution (50X) | 25243 | 1 ml | |
| FastScan™ ELISA Kit #35172 Positive Control Type 2 | 47385 | 1 ea | |
| Sealing Tape | 54503 | 1 ea |
*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.
Description
The FastScan™ Total LC3B ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of LC3B. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with LC3B in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of LC3B.
*Antibodies in kit are custom formulations specific to kit.
IMPORTANT: This FastScan™ ELISA Kit requires 4 washes at Step 6 of the protocol.
Specificity/Sensitivity
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 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 posttranslational modifications during autophagy (6-9). 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 (6-10). 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 (11).
- 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.
- Mann, S.S. and Hammarback, J.A. (1994) J. Biol. Chem. 269, 11492-97.
- Lang, T. et al. (1998) EMBO J. 17, 3597-607.
- Kabeya, Y. et al. (2000) EMBO J. 19, 5720-28.
- He, H. et al. (2003) J. Biol. Chem. 278, 29278-87.
- Tanida, I. et al. (2004) J. Biol. Chem. 279, 47704-10.
- Wu, J. et al. (2006) Biochem. Biophys. Res. Commun. 339, 437-42.
- Ichimura, Y. et al. (2000) Nature 408, 488-92.
- Kabeya, Y. et al. (2004) J. Cell Sci. 117, 2805-12.
Background References
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
Trademarks and Patents
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