Triple tandem FLAG epitope tag (3xFLAG)
Three tandem FLAG epitopes, 22 residues encoding DYKDHDGDYKDHDIDYKDDDDK, made of two DYKDHD variants and the original FLAG sequence DYKDDDDK. The extra epitopes lower the detection limit about tenfold versus a single FLAG in anti-FLAG M2 western blots, so the tag suits proteins expressed at low levels.
Origin: Synthetic oligonucleotide cassette of three tandem FLAG epitopes
Characteristics
Encodes DYKDHDGDYKDHDIDYKDDDDK, 22 amino acids in 66 nucleotides. The two DYKDHD variants add M2 antibody binding sites without adding enterokinase sites, leaving the single DDDDK site at the C-terminal end. In an M2 western blot, 3xFLAG-tagged alkaline phosphatase was detected at tenfold lower amounts than a single-FLAG fusion, down to 500 pg. The sequence favors M2 over M5 or M1. Enterokinase cuts after the lysine, so an N-terminal 3xFLAG can be cleaved off, leaving no residue on the cargo.
Applications: Detection by anti-FLAG M2 western blot and by M2-HRP immunostaining of fixed cells, and affinity purification on anti-FLAG M2 resin, for proteins expressed from a plasmid in COS-7 cells or in E. coli. Suited to proteins expressed at low levels.
Limitations: Enterokinase removal works only from an N-terminal position, so a C-terminal or internal 3xFLAG stays on the protein. At 22 residues and 66 nucleotides the tag is nearly three times longer than the eight-residue single FLAG, which adds to the coding payload of a fusion.
Sequence
References
- Hernan et al. (2000). Multiple epitope tagging of expressed proteins for enhanced detection. Biotechniques - Hernan 2000 3xFLAG Multiple Epitope Tagging