FLAG-tag
Encyclopedia
FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag
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 wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits.
A FLAG-tag can be used in many different assays
that require recognition by an antibody
. If there is no antibody against the studied protein, adding a FLAG-tag to this protein allows one to follow the protein with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence
or detection by SDS PAGE protein electrophoresis.
The peptide sequence of the FLAG-tag is: N-DYKDDDDK-C (1012 Da). It can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag
. It can be fused to the C-terminus or the N-terminus of a protein. Some commercially available antibodies (e.g., M1/4E11) recognize the epitope
only when it is present at the N-terminal. However, other available antibodies (e.g., M2) are position-insensitive.
The FLAG-tag was the first example of a fully functional epitope tag to be published in the scientific literature
and, being the first, was the only epitope tag to be patented. Unlike some other tags (e.g. myc, HA), where a monoclonal was first isolated against an existing protein, then the epitope was characterized and used as a tag, the FLAG epitope was designed first, and then monoclonals were raised to recognize it. The FLAG tag's structure has been optimized for compatibility with the proteins it is attached to, in that it is more hydrophilic than other common epitope tags and therefore less likely to denature or inactivate proteins to which it 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
).
The first published report of another epitope tagging method (HA-tag) appeared about one year after the Flag system had been sent to laboratories throughout the world for beta-testing as a kit for recombinant protein production.
Epitope tagging studies have become so widespread in modern molecular biology research that every major disease has been studied with epitope tagging techniques.
Protein tag
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes....
that can be added to a protein using recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
technology
Technology
Technology is the making, usage, and knowledge of tools, machines, techniques, crafts, systems or methods of organization in order to solve a problem or perform a specific function. It can also refer to the collection of such tools, machinery, and procedures. The word technology comes ;...
. It can be used for affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
, then used to separate recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits.
A FLAG-tag can be used in many different assays
Bioassay
Bioassay , or biological standardization is a type of scientific experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants...
that require recognition by an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
. If there is no antibody against the studied protein, adding a FLAG-tag to this protein allows one to follow the protein with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence
Immunofluorescence
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows...
or detection by SDS PAGE protein electrophoresis.
The peptide sequence of the FLAG-tag is: N-DYKDDDDK-C (1012 Da). It can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag
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 wild type protein expressed by the host organism...
. It can be fused to the C-terminus or the N-terminus of a protein. Some commercially available antibodies (e.g., M1/4E11) recognize the epitope
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 part of an antibody that recognizes the epitope is called a paratope...
only when it is present at the N-terminal. However, other available antibodies (e.g., M2) are position-insensitive.
The FLAG-tag was the first example of a fully functional epitope tag to be published in the scientific literature
and, being the first, was the only epitope tag to be patented. Unlike some other tags (e.g. myc, HA), where a monoclonal was first isolated against an existing protein, then the epitope was characterized and used as a tag, the FLAG epitope was designed first, and then monoclonals were raised to recognize it. The FLAG tag's structure has been optimized for compatibility with the proteins it is attached to, in that it is more hydrophilic than other common epitope tags and therefore less likely to denature or inactivate proteins to which it 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
Enteropeptidase is an enzyme produced by cells of the duodenum and involved in human digestion. It is secreted from intestinal glands following the entry of ingested food passing from the stomach...
).
The first published report of another epitope tagging method (HA-tag) appeared about one year after the Flag system had been sent to laboratories throughout the world for beta-testing as a kit for recombinant protein production.
Epitope tagging studies have become so widespread in modern molecular biology research that every major disease has been studied with epitope tagging techniques.