Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using _CG‐SLENP

This article describes a robust method for dual labeling of newly synthesized proteins and existing proteins. Assessing the properties of individual newly synthesized proteins from the bulk proteome is challenging due to difficulty in specifically isolating them. Previous methods such as non-natural amino acid labeling, isotope-labeled amino acid labeling, and puromycin labeling are effective for ensemble measurements. However, these existing methods do not support live-cell tracking or dynamic studies for a specific target protein. We designed a chemical genetics-based method for selective labeling of existing and newly synthesized proteins (_CG-SLENP). Using nuclear lamin A (LA) tagged with a HaloTag (HaloTag-LA) as an exemplar protein and various Halo ligands, we describe _CG-SLENP for labeling existing proteins and newly synthesized proteins. This approach can label these proteins either one at a time or dually in the same live cell. This method holds great potential for broader applications to study any given protein of interest.©2025 Wiley Periodicals LLC. Basic Protocol 1: _CG-SLENP labeling using clickable Halo ligands Basic Protocol 2: _CG-SLENP labeling using fluorescent Halo ligands.