Revvity, 64LC3B2PEG, HTRF Human and Mouse LC3B II Detection Kit, 500 Assay Points

Overview
LC3 is a mammalian homolog of the yeast ATG8 protein and is distributed throughout mammalian tissues and cultured cells. LC3 is a key component of the autophagy process - a degradative program that maintains cellular homeostasis.
During autophagy, cytoplasmic components such as cytosolic proteins and organelles are engulfed by autophagosomes Concurrently, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II). This newly formed conjugate is recruited to autophagosomal membranes. The subsequent fusion of autophagosomes with lysosomes results in the formation of autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II within the autolysosomal lumen is degraded.
The LC3-II level has been found to be proportional to autophagosome abundance making LC3-II the most popular marker of autophagy.
How it works
LC3B-II assay principle
The HTRF LC3B-II assay quantifies the expression level of LC3B-II in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The LC3B-II assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of LC3B-II in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

LC3B-II two-plate assay protocol
The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the LC3B-II HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

LC3B-II one-plate assay protocol
Detection of LC3B-II with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS-designed protocol enables miniaturization while maintaining robust HTRF quality. Application-Protein Quantification
Brand-HTRF
Detection Modality-HTRF
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Biomarkers
Target Species-Human Mouse
Technology-TR-FRET
Unit Size-500 assay points