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Autophagy is one of the major protein degradation machineries responsible for the breakdown and turnover of abnormal proteins and damaged organelles to maintain cellular homeostasis. Autophagy plays an important role in degradation of key misfolded proteins involved in the pathogenesis of neurodegenerative disorders. Dysfunction in the autophagy pathway impedes the clearance of aggregate-prone targets, and has been associated with accelerated disease progression. Furthermore, impairment in the maturation of autophagosomes to autolysosomes, resulting in the accumulation of autophagic vacuoles and autophagy-related vesicular compartments, has been observed in neurodegenerative disorders.

 

The Autophagy Antibody Sampler Kit offers antibodies for protein modifiers (e.g. ubiquitin), autophagy initiators (e.g. beclin-1), and proteins responsible for extension and maturation of autophagosomes (e.g. ATG5, LC3, and Rab7A). The components of this kit are each essential for evaluating autophagy or protein modification under normal and pathological conditions.

 

Autophagy Antibody Sampler Kit contents:

Specificity

Clone

Format

Reactivity

Application

Ubiquitin

P4G7

Purified

Extensive (Yeast to Hu)

IHC-P, WB

Rab7A

W16034A

Purified

Hu, Ms, Rat

IHC-P, ICC, WB, Direct ELISA

Beclin-1

O93F3

Purified

Hu, Ms, Rat

WB

ATG5

177.19

Purified

Hu, Ms, Rat

IHC-P, ICC, WB

LC3

A15143K

Purified

Hu, Ms

IHC-P, ICC

Anti-ubiquitin antibodies are commonly used in immunohistochemistry to detect proteinaceous inclusions that are formed in a variety of neurodegenerative diseases, as ubiquitinated species of misfolded proteins are the major constituents of these inclusions and often accumulate in the cytosol. Clone P4G7 is ideal for detection of free or monomeric ubiquitin as well as polyubiquitinated proteins.

 

 

Anti-ATG5 antibodies are utilized as a marker for newly formed phagophores, also referred to as isolation membranes. Clone 177.19 detects ATG5 by IHC, ICC, and western blotting.

 

 

 

LC3 is initially cleaved by ATG4 to generate LC3-I, which is subsequently converted to LC3-II as a result of conjugation to phosphatidylethanolamine by the ATG5 complex. LC3-II remains associated with autophagosomes and hence is the most common marker for detection of these structures.

 

 

 

Rab7, a small GTPase of the Rab family, plays roles in vesicular transport, endocytic trafficking, and autophagy. Rab7 associates with late endocytic structures and can be used as a marker for late endosomes.

 

 

 

Beclin-1 is part of the PI3K complex that facilitates the generation of phosphatidylinositol 3-phosphate and the formation of phagophores. Beclin-1 can block or induce autopahgy depending on which proteins it is interacting with. Antibodies against beclin-1 are used to detect its expression levels, subcellular localization, and binding partners.

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