Fluorescence-based Screening of Incorporation Efficiencies
4:22
Genetic Incorporation of ncAAs into GPCRs
8:21
Ultrafast Bioorthogonal Labeling of GPCRs on Live Mammalian Cells
11:11
Results: Incorporation of Chemical Probes into GPCRs in Mammalian Cells for Cross-linking and Imaging Studies
12:52
Conclusion
Transcription
The overall goal of this protocol is to enhance the incorporation rate of non-canonical amino acids in mammalian cells to tackle biological questions in live cell settings including mapping of protein-protein interaction surfaces, and bioorthogona
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A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.