Fluorescent bioorthogonal labeling of class B GPCRs in live cells.

ABSTRACT: In this method paper, we describe the protocols for selective labeling of GCGR, a member of the class B GPCR family regulating glucose homeostasis, in live cells. A two-step procedure is presented in which a strained alkene chemical reporter is inserted into any desired location within the GPCR in the first step, followed by a robust bioorthogonal ligation reaction with a fluorophore-conjugated tetrazine or tetrazole reagent in the second step. The amber codon suppression strategy was adopted for site-specific incorporation of the strained alkene reporter, either spirohexene or trans-cyclooctene, in HEK293T cells. Subsequently, the inverse electron-demand Diels-Alder reaction with an AF647-conjugated 3,6-di (2-pyridyl)-S-tetrazine (DpTz) was performed with the alkene-encoded GCGR on live-c

SUBMITTER: Gangam SK 

PROVIDER: S-EPMC7643056 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature