Titlebook: Genetically Incorporated Non-Canonical Amino Acids; Methods and Protocol Yu-Hsuan Tsai,Simon J. Els?sser Book 2023 The Editor(s) (if applic

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https://doi.org/10.1007/978-1-349-16014-3fficiency, which can be quantitatively converted into an acyl hydrazide via hydrazinolysis. Then, through a reaction with acetyl acetone, the acyl hydrazide is converted to reactive Knorr pyrazole. Finally, the in situ generated Knorr pyrazole is incubated with methylamine to give Gln methylation.

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Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incors biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.

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Engineering Homogeneous Photoactive Antibody Fragmentseplacement with photocaged tyrosine (pcY). This is followed by cloning of plasmids and expression of pcY-containing antibody fragments in .. Finally, we describe a cost-effective and biologically-relevant method for measuring the binding affinity of photoactive antibody fragments to antigens expressed on the surface of live cancer cells.

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Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the?Analysis of Deubiqminocoumarin-derived fluorescent ncAA into proteins in . and preparation of a fluorescent ncAA-based FRET probe for assaying the?activities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.

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