As a protein tag affinity tag or epitope tag (german for tag, label ',' labels ' or ' label ') are in biochemistry different, mostly short, designated amino acid sequences, can be labeled with the help of proteins. The labeled proteins belong to the fusion proteins.
The various markers are differently suitable for different purposes. They are in the course of protein design in the production of recombinant proteins and their purification and detection of the affinity, via pull-down assays by Western blot, by immunohistochemistry, used by fluorescence microscopy or in live imaging.
To produce a protein tag, the DNA sequence encoding the protein tag is inserted to give the reading frame in the DNA sequence encoding the fusion protein after the start codon and before the stop codon. Wherein an N-terminal or a C-terminal protein tag is formed on the protein during translation.
Occasionally, the protein -tag of the protein has to be removed after purification, which may be accomplished by a protease cleavage site, or an inducible intein eg. The proteolysis -based approach uses proteases with longer recognition sequence, the possible overlap only at the interface of protein tags, such as the TEV protease. Inteins can be induced by thiols or by lowering the pH.
Some tags have not only a binding of an antibody or immunoconjugate of other functions such as:
- Immunaffinitätschromatografische purification (all with the corresponding antibodies )
- Antibody -based evidence (at all, for example, by Western blot, immunohistochemistry or immunofluorescence )
- Affinity chromatography because of the affinity to any other binding partner (e.g., CaM tag, CBP -tag, GST-tag, MBP -tag)
- Biotinylation for purification with streptavidin (eg Avi-tag, BCCP tag, Strep-tag )
- Complex formation with divalent nickel ions and nitrilotriacetic acid -modified and cross-linked agarose or dextran (eg His-tag )
- Fluorescent label (such as GFP or flash day)
- Easier separation by HPLC due to the increased polarity (for example, His tag, FLAG tag, Xpress -tag)
- Cysteine as reactive groups (for example, GST-tag, thioredoxin tag) or enzymatic self- modification ( snap- day),
- Increasing the solubility in inclusion bodies with larger tags ( e.g. GST-tag, MBP -tag)
- Improvement of protein folding in inclusion bodies or due fehlverknüpfter disulfide bridges (eg thioredoxin tag, poly (NANP ) tag )
- Inducible precipitation reactions