Journal: Nucleic Acids Research

Article Title: HaloTag-based conjugation of proteins to barcoding-oligonucleotides

doi: 10.1093/nar/gkz1086

Figure Lengend Snippet: HaloTag-based protein barcoding. ( A ) Schematic diagram of the amine-modified barcode DNA. ( B ) The HaloTag-based protein barcoding assay. Plasmids harboring specific ORFs fused with HaloTag are first prepared. Addition of a coupled transcription-translation reagent results in the expression of a HaloTag fusion ORF protein. The HaloTag protein binds covalently to ester–chloroalkane ligand linked to an amido-modified oligonucleotide. The method enables protein molecule detection using the DNA barcode.

Article Snippet: The barcoded proteins were treated with HaloTag ligand (Promega) at RT for 1 h to fill the empty HaloTag ligand capture sites; 25 μl bait protein (pIX-His-HALO-ORFs) produced using the TNT-coupled wheat germ extract (Promega) was mixed with 5 μl Magne HaloTag beads and incubated at RT for 1 h. The beads coupled with bait proteins were then washed with PBS and mixed with 5 μl barcoded proteins at RT for 2 h. The mixture was next washed 3 times with PBS and used for library preparation for NGS.

Techniques: Modification, Expressing