Peptide-Tag Specific Nanobodies for Studying Proteins in Live Cells.

Single-domain antibodies such as nanobodies (Nbs) have substantially expanded the possibilities of advanced cellular imaging. In comparison to conventional antibodies, Nbs are characterized by small size, high stability, and solubility in many environments, including the cytoplasm. Nbs can be efficiently functionalized or modified according to the needs of the imaging approach. Target-specific Nbs can be easily converted into genetically encoded fluorescently labeled intrabodies, also known as chromobodies (CBs), which represent powerful tools to study the dynamics of different proteins of interest within living cells. In this context, CBs specific for a short peptide epitope provide a versatile alternative to bypass the limitations observed with larger fluorescent protein fusions and can be readily used to visualize and monitor peptide-tagged proteins for which specific Nbs are not available. Here, we present our novel detection system comprising a 15 amino acid peptide-tag (PepTag) in combination with a peptide-tag specific CB (PepCB). We provide protocols for adding the PepTag to different proteins of interest, reformatting the peptide-specific Nb (PepNb) into a CB for expression in mammalian cells, and establishment of stable cell lines expressing the PepCB for protein interaction assays and compound screenings.