Scholarlink

<p>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 label a neuromodulatory receptor with dyes optimized for stimulated emission by depletion super-resolution microscopy, allowing accuracy in distinguishing surface versus internal receptors of the presynaptic terminal. This approach offers broad utility for surface-specific protein labelling.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月12日

查看全文
  • Kilian Roßmann,
  • Ulrich Pabst,
  • Bianca C. Baciu,
  • Siqi Sun,
  • Christiane Huhn,
  • Christina Holmboe Olesen,
  • Maria Kowald,
  • Eleni Tapp,
  • Marie Bieck,
  • Ramona Birke,
  • Brenda C. Shields,
  • Pyeonghwa Jeong,
  • Jiyong Hong,
  • Michael R. Tadross,
  • Joshua Levitz,
  • Martin Lehmann,
  • Noa Lipstein,
  • Johannes Broichhagen

摘要

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 label a neuromodulatory receptor with dyes optimized for stimulated emission by depletion super-resolution microscopy, allowing accuracy in distinguishing surface versus internal receptors of the presynaptic terminal. This approach offers broad utility for surface-specific protein labelling.