Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag...

Area of Science:

• Biochemistry
• Cell Biology
• Microscopy

Background:

• Self-labeling protein tags are crucial for fluorescent protein visualization in optical microscopy.
• Dye membrane permeability and background signals hinder extracellular labeling analysis.

Purpose of the Study:

• To develop an improved method for extracellular labeling of SNAP-tagged proteins.
• To reduce non-specific staining and background noise in fluorescence microscopy.

Main Methods:

• Functionalization of SNAP-tag substrate benzyl guanine (BG) with a charged sulfonate (SBG).
• Development of SBG-conjugated fluorophores across the visible spectrum.
• Application in live-cell imaging, superresolution microscopy, and in vivo studies.

Main Results:

• SBG-conjugated fluorophores cleanly label SNAP-fused proteins on the plasma membrane.
• The SBG modification improves solubility and reduces non-specific staining.
• Successful interrogation of G protein-coupled receptors (GPCRs) using various imaging techniques.

Conclusions:

• SBG-functionalized substrates offer a robust solution for enhanced extracellular protein labeling.
• This method improves the accuracy and resolution of cellular imaging studies.
• The approach is versatile for studying membrane proteins like GPCRs in diverse biological contexts.