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Anti Autophagy antibodies

Background
Autophagy is the primary means for the degradation of cytoplasmic constituents in the lysosome and has been considered to be an important defensive mechanism during starvation. Three distinct types of autophagy have been identified, macroautophagy, microautophagy and chaperone-mediated autophagy, and, among these, the molecular mechanism and physiologic significance of macroautophagy have been best studied. Macroautophagy (usually referred to simply as autophagy) is mediated by the autophagosome, a unique and specialized cellular organelle. Atg5, Atg12 and Atg16L form a 800-kD protein complex in the cytosol. Almost all Atg5 and Atg12 are covalently attached to each other and present as an Atg12-Atg5 conjugate within cells. A small fraction of the Atg12-Atg5Atg16L complex targets to the outer side of isolation membrane (or phagophore), which is an intermediate structure during autophagosome formation. The protein complex plays an essential role in the membrane elongation and dissociate from the membrane upon completion of autophagosome formation. Recent Atg5 gene knockout studies have suggested that autophagy is important for a variety of physiological processes such as starvation adaptation during food withdrawal or after birth, intracellular protein quality control and killing of intracellular pathogens..


Fig1. Intracellular protein degradation systems
Macroautophagy. A small volume of cytoplasm is enclosed by the autophagic isolation membrane, which eventually results in the formation of an autophagosome. The outer membrane of the autophagosome then fuses with the lysosome where the cytoplasm-derived materials are degraded.

Antibody to Autophagy marker proteins

LC3

LC3B is one of the mammalian Atg8 homologs and widely used as an autophagosome marker. Immediately after synthesis, LC3 is processed by Atg4 and becomes LC3-I. Upon induction of autophagy, the C-terminal glycine of LC3-I is conjugated to phosphatidylethanolamine, resulting in formation of membrane-bound LC3-II. Most LC3-II is thought be present on autophagosome membrane. The autophagosome subsequently fuses with a lysosome, where inside materials, including LC3-II, are degraded. The expression level of LC3-II generally correlates with the number of autophagosome.

#Clone LC3-1703
Monoclonal antibody LC3-1703 is designed for application of immunocytochemistry. As amount of LC3-II positively correlates to autophagozome formation, this antobody is useful tool for autophagy detection in various cells (Fig2). This is applicable to cells of Human or Mouse and and some examples are found applying this to immunoelectron microscopy (Fig3).


(Fig2)Immunofluorescence microscopy analysis of mouse embryonic fibroblasts (MEFs) with #LC3-1703


(Fig3) Immuno-electron microscopy analysis of mouse embryonic fibroblasts (MEFs) with #LC3-1703

#Clone LC3.No.6
Clone LC3.No.6 reacts both with LC3-I and LC3-II and suitable for detection of each formation individually by western blot (Fig4).


(Fig4) Immunoblot for LC3B with #LC3.No.6

Atg7
Autophagy is an evolutionaly conserved machinery, in which autophagosome fuses with lysosome and degrades bulk cytoplasmic contents. Autophagy is involved in many physiological processes such as development, infection, cancer, and neurodegenerative diseases. ATG (autopha gy-related) genes were identified by genetic screening in yeast. Atg7 acts as an E1-like enzyme in both Atg12 and Atg8 ubiquitin-like conjugation systems. Atg7 transfers Atg12 to an E2-like enzyme Atg10, and conjugates Atg12 to Atg5. In the other hand, Atg7 transfers Atg8 to another E2-like enzyme Atg3, and conjugates Atg8 to phosphatidylethanolamine. Many of these ATG genes are conserved also in mammals. Atg7 deficient neonates die soon after birth as they cannot endure perineonatal starvation. Conditional deletion of Atg7 in nerves system results in neurodegeneration with ubiquitin containing aggregates.



BIF1
Bif1 (Bax interacting factor 1; also known as SH3GLB1 was initially cloned as a binding partner of the pro-apoptotic Bax protein. This protein is a member of the endophilin B protein family and contains an amino-terminal N-BAR domain and a carboxy-terminal Src homology 3 (SH3) domain1), . Bif1 gene encodes a predicted protein of 365 amino acid and mouse and human Bif-1 protein are highly conserved and share 96% identity at the amino acids level. RNA expression of Bif-1 in human tissue is abundant in heart, skeletal muscle, kidney, and placenta1). Bif-1 protein plays a critical role in apoptosis, mitochondrial morphology and autophagy. Loss of Bif-1 suppresses programmed cell death and promotes tumorigenesis5).



Gtr2
Gtr2 (341 amino acids, 38.6 kDa) is the yeast homologue of RagC identified in yeast and classified as a Ras-like small GTPase subfamily. In cytoplasm, GTP-bound Gtr2 forms a heterodimer with Gtr1, which is involved in autophagy through mTOR signal pathway.



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