AlphaLISA® no-wash immunoassay kit for detection of human LC3B in buffered solution or cell lysates. The antibodies in the kit detect both LC3B type I and type II. The analyte in this kit consists of recombinant LC3B fused to His-tag at the N-terminus.
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LC3 represents a mammalian homologue of the yeast autophagy related gene ATG8. It was originally characterized as light chain 3 of the microtubule associated protein 1 (MAP1LC3). The protein family consists of LC3 A, B, and C and the GABARAP subfamily. Human LC3B is 125 amino acids long. After synthesis, it is cleaved by ATG4B to expose a C-terminal glycine, representing the cytosolic form LC3B I. During autophagy the C-terminus is covalently linked to autophagosomal vesicle membranes via a phospholipid anchor and this form is called LC3B II. The transformation of LC3B I to II is mediated by a ubiquitination-like process involving ATG7 (E1), ATG3 (E2) and the ATG16L complex (E3). To date, LC3B is considered as the most persistent marker of the autophagy pathway.
AlphaLISA technology allows the detection of molecules of interest in a no-wash, highly sensitive, quantitative assay. In an AlphaLISA assay, a biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads causes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.
|Assay Target Class||Protein|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||500 assay points|
The aim of this work was to compare the performance of the AlphaLISA® amplified luminescent proximity homogeneous assay technology platform and an alternative time-resolved fluorescenceresonance energy transfer (TR-FRET) technology assay platform for the detection of five biomarkers.
Biomarker levels were measured directly in CulturPlates-96 and-384 in a simple, fast, all-in-one-well AlphaLISA assay format. The elimination of transfer and wash steps simplifies cellular assays, reduces variability and significantly reduces hands-on time and costs associated with consumables. Integral plasma membrane (EGFR) and secreted solubleproteins (TNFa, IL1ß, IL6, IL8) were successfully determined, on adherent or suspension cells, using the standard AlphaLISA Immunoassay buffer.
The AlphaLISA® assay is a homogeneous immunoassay alternative to classical ELISA. AlphaLISA assays were originally utilized to detect analytesin cell cultures upernatants or serum/plasma samples.
Enzyme-linked Immunosorbent Assay is the most widely Kits adopted method for detection and quantification of cytokines and other biomarkers. This traditional technology offers good,selectivity, sensitivity and assay versatility; however, it has certain disadvantages such as limited dynamic range and low throughput due to the numerous wash steps.