|17208C||1 Kit (96 assays)||
|Product Includes||Volume||Solution Color|
|Atg13 Rabbit mAb Coated Microwells||96 tests|
|Phospho-Atg13 (Ser355) Rabbit Detection mAb||1 ea||Red (Lyophilized)|
|HRP Diluent||5.5 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
The rapid protocol (RP) PathScan® RP Phospho-Atg13 (Ser355) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Atg13 protein when phosphorylated at Ser355 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 phospho-Atg13 (Ser355) 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 phospho-Atg13 (Ser355). Learn more about all of your ELISA kit options here.
*Antibodies in this kit are custom formulations specific to kit.
NOTE: This protocol is for PathScan® kits that use an HRP directly conjugated to the detection antibody (Rapid Protocol), rather than a 2-step method where the detection antibody and a secondary-HRP are added sequentially.
NOTE: Prepare solutions with deionized/purified water or equivalent.
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
created July 2020
Protocol Id: 2144
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.
Atg13/Apg13 was originally identified in yeast as a constitutively expressed protein that was genetically linked to Atg1/Apg1, a protein kinase required for autophagy (4). Overexpression of Atg1 suppresses the defects in autophagy observed in Atg13 mutants (4). Autophagy requires a direct association between Atg1 and Atg13, and is inhibited by TOR-dependent phosphorylation of Atg13 under high-nutrient conditions (5). Similarly, mammalian Atg13 forms a complex with the Atg1 homologues ULK1/2, along with FIP200, which localizes to autophagic isolation membranes and regulates autophagosome biogenesis (6-8). mTOR phosphorylates both Atg13 and ULK1, suppressing ULK1 kinase activity and autophagy (7-9). ULK1 can directly phosphorylate Atg13 at a yet unidentified site, presumably to promote autophagy (7,8). Additional studies suggest that Atg13 and FIP200 can function independently of ULK1 and ULK2 to induce autophagy through an unknown mechanism (10).
ULK1-dependent phosphorylation of Atg13 at Ser355, which corresponds to Ser318 of isoform 2 of Atg13, leads to the recruitment of Atg13 to damaged mitochondria, enabling efficient mitophagy (11).
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