What is biotin phenol?
APEX is an ascorbate peroxidase derivative reliant on hydrogen peroxide for catalyzing the oxidation of biotin-tyramide, also known as biotin-phenol, to a short-lived and reactive biotin-phenol free radical. Labeling is achieved when this intermediate reacts with various functional groups of nearby biomolecules.
What is Apex tagging?
In 2012, the APEX method was first introduced as a genetic tag for the high-resolution imaging of cellular ultrastructures. (16) APEX, as a newly developed genetic tag, catalyzes the H2O2-dependent oxidative polymerization of 3′-diaminobenzidine (DAB) to generate a localized precipitate, similar to the function of HRP.
What is APEX2?
APEX2 is an engineered form of a soybean ascorbate peroxidase (APX1). It can be used as a genetically encoded tag for electron microscopy (EM) or as a proximity labeling tool for proteomic analysis. For EM, cells expressing APEX2 are fixed and then exposed to diaminobenzidine (DAB) and hydrogen peroxide.
What is TurboID?
TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels proximal proteins.
How do you Biotinylate a protein?
coli biotin holoenzyme synthetase, also known as biotin ligase (BirA, P06709). The most common way of targeting a protein of interest is by fusing the protein at its N-terminus, C-terminus or at an internal loop to a 15 amino acid peptide ( GLNDIFEAQKIEWHE ), termed AviTag or Acceptor Peptide (AP).
What is the protein apex?
APEX Grass-Fed Whey Protein contains only whole intact whey protein concentrate with no amino acid spiking, no banned substances, no artificial colours, fillers or aspartame. Each serving contains 30 grams of grass-fed whey protein concentrate that is low in carbs and contains no trans fats.
What is BioID?
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. Proteins identified by BioID are candidate interactors for the protein of interest.
How does streptavidin bind biotin?
Streptavidin is a tetramer and each subunit binds biotin with equal affinity. Multivalency is an advantage in applications like MHC tetramer staining, where avidity effects improve the ability of MHC molecules attached to streptavidin to detect specific T cells.
How much biotin is in each antibody?
Biotin is used in two-step detection systems in concert with conjugated avidin. Biotin is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 3 and 6 biotin molecules are conjugated to each antibody.
How is apex used in biotinylation of proteins?
In the presence of H2O2, engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are subsequently enriched by streptavidin and identified by mass spectrometry.
How is APEX2 used to identify protein interactions?
The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein-protein interactions. APEX2 fused to a protein of interest covalently tags nearby proteins with biotin-phenol (BP) when H2O2 is added to the cell culture medium. Subsequent affinity purif …
How does apex catalyze oxidation of biotin phenol?
APEX catalyzes the oxidation of biotin-phenol to generate a very short-lived radical, which tags proteins proximal to APEX, allowing the proteins closest to the genetically labeled protein to be identified by mass spectrometry.
How is APEX2 tool used in proteomic analysis?
The APEX2 tool enables proteomic mapping of subcellular compartments as well as identification of dynamic protein complexes, and has emerged as a new methodology for proteomic analysis. Despite these advantages, a related APEX2 approach has not been developed for yeast.