A general one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging.

ABSTRACT: Visualization of proteins can be achieved by genetically grafting HaloTag Protein (HTP) into the protein of interest followed by incubation with a dye-linked HaloTag Ligand (HTL). This approach allows for use of fluorophores optimized for specific optical techniques or of cell-impermeable dyes to selectively label cell surface proteins. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Here we show that several dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed, using a one-step protocol. We validate such compounds, termed dye-SHTL (dye shuttle), in living cells, and demonstrate exclusive membrane staining. In transduced primary hippocampal neurons, we la

SUBMITTER: Roßmann K 

PROVIDER: S-EPMC12796469 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature