Revision 11
#71484
Store at +4C
PathScan® RP Phospho-Atg14 (Ser29) Sandwich ELISA Kit
1 Kit
(96 assays)
Species Cross Reactivity:
H
UniProt ID:
#Q6ZNE5
Entrez-Gene Id:
#22863
877-616-CELL (2355)
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes | Product # | Quantity | Color | Storage Temp |
---|---|---|---|---|
Atg14 Rabbit mAb Coated Microwells | 37097 | 96 tests | +4C | |
Phospho-Atg14 (Ser29) Rabbit Detection mAb | 57676 | 1 ea | Red (Lyophilized) | +4C |
HRP Diluent | 13515 | 5.5 ml | Red | +4C |
TMB Substrate | 7004 | 11 ml | +4C | |
STOP Solution | 7002 | 11 ml | +4C | |
Sealing Tape | 54503 | 2 ea | +4C | |
ELISA Wash Buffer (20X) | 9801 | 25 ml | +4C | |
Cell Lysis Buffer (10X) | 9803 | 15 ml | -20C |
*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.
Description
The rapid protocol (RP) PathScan® RP Phospho-Atg14 (Ser29) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Atg14 protein phosphorylated at Ser29 in a reduced assay time of 1.5 hours. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with Atg14 protein phosphorylated at Ser29 in a single step. The plate is then extensively washed and TMB reagent is added for signal development. The magnitude of absorbance for the developed color is proportional to the quantity of Atg14 protein phosphorylated at Ser29. Learn more about all of your ELISA kit options here.
*Antibodies in this kit are custom formulations specific to kit.
Specificity/Sensitivity
The PathScan® RP Phospho-Atg14 (Ser29) Sandwich ELISA Kit detects endogenous levels of Atg14 protein phosphorylated at Ser29. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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 is also 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 is directed by a number of autophagy-related (Atg) genes. These proteins are involved in the formation of autophagosomes, cytoplasmic vacuoles that are delivered to lysosomes for degradation. The class III type phosphoinositide 3-kinase (PI3K) Vps34 regulates vacuolar trafficking and autophagy (4,5). Multiple proteins associate with Vps34, including p105/Vps15, Beclin-1, UVRAG, Atg14, and Rubicon, to determine Vps34 function (6-12). Atg14 and Rubicon were identified based on their ability to bind to Beclin-1 and participate in unique complexes with opposing functions (9-12). Rubicon, which localizes to the endosome and lysosome, inhibits Vps34 lipid kinase activity; knockdown of Rubicon enhances autophagy and endocytic trafficking (11,12). In contrast, Atg14 localizes to autophagosomes, isolation membranes and ER, and can enhance Vps34 activity. Knockdown of Atg14 inhibits starvation-induced autophagy (11,12).
The serine/threonine kinase ULK1 phosphorylates Atg14 at Ser29 to promote autophagosome formation (13).
Background References
- 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.
- Corvera, S. (2001) Traffic 2, 859-66.
- Yan, Y. and Backer, J.M. (2007) Biochem Soc Trans 35, 239-41.
- Stack, J.H. et al. (1995) J Cell Biol 129, 321-34.
- Zeng, X. et al. (2006) J Cell Sci 119, 259-70.
- Liang, C. et al. (2006) Nat Cell Biol 8, 688-99.
- Itakura, E. et al. (2008) Mol Biol Cell 19, 5360-72.
- Sun, Q. et al. (2008) Proc Natl Acad Sci U S A 105, 19211-6.
- Zhong, Y. et al. (2009) Nat Cell Biol 11, 468-76.
- Matsunaga, K. et al. (2009) Nat Cell Biol 11, 385-96.
- Park, J.M. et al. (2016) Autophagy 12, 547-64.
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 G: Goat All: All Species Expected
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
Limited Uses
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
Revision 11
Figure 1. Atg14 is phosphorylated at Ser29 in 293T cells that were co-transfected with constructs expressing human Atg14 and mouse ULK1, but is not phosphorylated in cells transfected with only an Atg14 expression construct. The relationship between lysate protein concentration from Atg14/ULK1 co-transfected and Atg14-only transfected cells and the absorbance at 450 nm using the PathScan® RP Phospho-Atg14 (Ser29) Sandwich ELISA Kit #71484 is shown in the upper figure. The corresponding western blots using phospho-Atg14 (Ser29) antibody (left panel) and Atg14 antibody (right panel) are shown in the lower figure. 293T cells were either co-transfected with constructs expressing GFP-tagged human Atg14 protein (hAtg14-GFP) and mouse ULK1 (mULK1) or just transfected with a construct expressing GFP-tagged human Atg14 protein (hAtg14-GFP), and then lysed.