FLAG-tag , or FLAG octapeptide , or FLAG epitope , is a tag of a polypeptide protein that can be added to a protein using recombinant DNA technology, has a sequence motif DYKDDDDK (where D = aspartic acid, Y = tyrosine, and K = lysine). This is a specific artificial antigen, a high affinity monoclonal antibody that has been developed and can therefore be used for protein purification by affinity chromatography and can also be used to search for proteins in living cells. It has been used to separate recombinant proteins, overexpressed from wild-type proteins expressed by host organisms. It can also be used in isolation of protein complexes with some subunits, since light purification procedures tend not to disturb the complex. It has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination with X-ray crystallography.
A FLAG tag can be used in tests that require introduction by antibodies. If there are no antibodies to the given protein, adding the FLAG tag to the protein allows the protein to be studied with antibodies to the FLAG sequence. Examples are cellular cellularization studies by immunofluorescence or detection by SDS PAGE and Western blotting protein electrophoresis.
The peptide sequence of the FLAG tag from N-terminus to C-terminus is: DYKDDDDK (1012 Da). In addition, it can be used together, generally 3xFLAG peptide: DYKDHD-G-DYKDHD-I-DYKDDDDK (with end tag encoding cleavage site of enterokinase). It can coalesce with C-terminus or N-terminus proteins, or incorporated into proteins. Some commercially available antibodies (eg, M1/​​â € <4E11) recognize epitope only when present in N-terminus. However, other available antibodies (eg, M2) are not sensitive to position. The tyrosine residue in the FLAG tag can be sulphate, which may affect the introduction of antibodies from the FLAG epitope. The FLAG tag can be used in conjunction with other affinity marks, such as polyhistidine tags (His-tag), HA-tags or myc-tags.
Video FLAG-tag
History
The first use of epitope tagging was described by Munro and Pelham in 1984. The FLAG tag is the second example of a fully functional and refined epitope tag, published in scientific literature. and the only patented epitope tags. It has since become the most commonly used protein tag in laboratories around the world. Unlike some other tags (eg myc, HA), in which monoclonal antibodies were first isolated against the existing protein, the epitope is characterized and used as a tag, FLAG epitopes are an idealized artificial design, in which monoclonal antibodies are raised.. The FLAG tag structure is optimized for compatibility with embedded proteins, in which case it is more hydrophilic than other common epitope tags and therefore less likely to change the properties or disable the added protein. In addition, N-terminal FLAG tags can be removed easily from proteins after they are isolated, with treatment with specific proteases, enterokinase (enteropeptidase).
The third epitope tagging report, (HA-tag), appears about a year after the Flag system was first sent.
Maps FLAG-tag
See also
References
Source of the article : Wikipedia
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