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Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click(More)
A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins(More)
Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the(More)
The intracellular trafficking of drugs is critical to the efficacy of drugs that are susceptible to attack by lysosomal enzymes. It is therefore an important goal to design and synthesize molecules which can enhance the transport of endocytosed drugs from the endosomal compartments to the cytoplasm. The pH of an endosome is lower than that of the cytosol by(More)
Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce non-canonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells' own translational(More)
We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or(More)
A family of uniform periodic polypeptides has been prepared by bacterial expression of the corresponding artificial genes, with the objective of exploring the potential for control of supramolecular organization in genetically engineered protein-based polymeric materials. The repeating units of the polypeptides consist of oligomeric alanyl-glycine sequences(More)
The coiled-coil protein motif occurs in over 200 proteins and has generated interest for a range of applications requiring surface immobilization of the constituent peptides. This paper describes an investigation of the environment-responsive behavior of a monolayer of surface-immobilized artificial proteins, which are known to assemble to form coiled-coil(More)
Long nanotubes of fluid-lipid bilayers can be used to create templates for photochemical polymerization into solid-phase conduits and networks. Each nanotube is pulled from a micropipette-held feeder vesicle by mechanical retraction of the vesicle after molecular bonding to a rigid substrate. The caliber of the tube is controlled precisely in a range from(More)