FLAG-tag

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a peptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence DYKDDDDK (where D=aspartic acid, Y=tyrosine, and K=lysine).^[1] It is one of the most specific tags^[2] and it is an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by affinity chromatography and also can be used for locating proteins within living cells. FLAG-tag has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. FLAG-tag can also be used in the isolation of protein complexes with multiple subunits, because FLAG-tag's mild purification procedure tends not to disrupt such complexes. FLAG-tag-based purification has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography.

A FLAG-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against a given protein, adding a FLAG-tag to a protein allows the protein to be studied with an antibody against the FLAG-tag sequence. Examples are cellular localization studies by immunofluorescence, immunoprecipitation or detection by SDS PAGE protein electrophoresis and Western blotting.

The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (1012 Da). Additionally, FLAG-tags may be used in tandem, commonly the 3xFLAG peptide: DYKDHD-G-DYKDHD-I-DYKDDDDK (with the final tag encoding an enterokinase cleavage site). FLAG-tag can be fused to the C-terminus or the N-terminus of a protein, or inserted within a protein. Some commercially available antibodies (e.g., M1/4E11) recognize the epitope only when FLAG-tag is present at the N-terminus. However, other available antibodies (e.g., M2) are position-insensitive. The tyrosine residue in the FLAG-tag can be sulfated when expressed on certain secreted proteins, which can affect antibody recognition of the FLAG epitope.^[3] The FLAG-tag can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag.

^ Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Pat Cerretti D, Urdal DL, Conlon PJ (1988). "A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification". Bio/Technology. 6 (10): 1204–10. doi:10.1038/nbt1088-1204. S2CID 205272216.
^ "Cube Biotech". Cube Biotech. Retrieved 2020-02-13.
^ Hunter MR, Grimsey NL, Glass M (2016). "Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model". Scientific Reports. 6: 27316. Bibcode:2016NatSR...627316H. doi:10.1038/srep27316. PMC 4895180. PMID 27273047.

[Hopp_1988-1] Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Pat Cerretti D, Urdal DL, Conlon PJ (1988). "A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification". Bio/Technology. 6 (10): 1204–10. doi:10.1038/nbt1088-1204. S2CID 205272216.

[2] "Cube Biotech". Cube Biotech. Retrieved 2020-02-13.

[Hunter_2016-3] Hunter MR, Grimsey NL, Glass M (2016). "Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model". Scientific Reports. 6: 27316. Bibcode:2016NatSR...627316H. doi:10.1038/srep27316. PMC 4895180. PMID 27273047.

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