FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a peptide

protein tag Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or a ...

that can be added to a protein using

recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fo ...

technology Technology is the application of knowledge to reach practical goals in a specifiable and Reproducibility, reproducible way. The word ''technology'' may also mean the product of such an endeavor. The use of technology is widely prevalent in me ...

, having the sequence DYKDDDDK (where D= aspartic acid, Y=

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...

, and K= lysine). It is one of the most specific tags 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 Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...

and also can be used for locating proteins within living cells. FLAG-tag has been used to separate recombinant, overexpressed protein from

wild-type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...

protein expressed by the host organism. FLAG-tag can also be used in the isolation of

protein complexes A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multienzyme complexes, in which multiple catalytic domains are found in a single polypeptide chain. Protein ...

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 X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...

. A FLAG-tag can be used in many different

assays An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...

that require recognition by an

antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

. 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 western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

. 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 An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitope is the specific piece of the antigen to which an antibody binds. The p ...

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. The FLAG-tag can be used in conjunction with other affinity tags, for example a polyhistidine tag (

His-tag A polyhistidine-tag is an amino acid motif in proteins that typically consists of at least six histidine (''His'') residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag, by the US tra ...

HA-tag Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human influenza virus. The HA-tag is derived from the HA-molecule corresponding to amino acids 98-106. HA-tag has been extensively used as a general ...

myc-tag A myc tag is a polypeptide protein tag derived from the c-myc gene product that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wil ...

History

The first use of

tagging was described by Munro and Pelham in 1984. The FLAG-tag was the second example of a fully functional, improved epitope tag, published in the scientific literature. and was the only epitope tag to be patented. It has since become one of the most commonly used protein tags in laboratories worldwide. Unlike some other tags (e.g. myc, HA), where a monoclonal antibody was first isolated against an existing protein, then the epitope was characterized and used as a tag, the FLAG epitope was an idealized, artificial design, to which monoclonal antibodies were raised. The FLAG-tag's sequence was optimized for compatibility with proteins it is attached to, in that FLAG-tag is more hydrophilic than other common epitope tags and therefore less likely to reduce the activity of proteins to which FLAG-tag is appended. In addition, N-terminal FLAG tags can be removed readily from proteins once they have been isolated, by treatment with the specific protease, enterokinase (

enteropeptidase Enteropeptidase (also called enterokinase) is an enzyme produced by cells of the duodenum and is involved in digestion in humans and other animals. Enteropeptidase converts trypsinogen (a zymogen) into its active form trypsin, resulting in the ...

). The third report of epitope tagging, (

), appeared about one year after the Flag system had been first shipped.

References

{{Protein tag Biochemical separation processes Biochemistry detection methods Laboratory techniques Molecular biology Peptide sequences

History

See also

References