FLAG-tag, or FLAG octapeptide, or FLAG epitope, is the first epitope tag designed for fusion proteins and is the only patented tag. The molecular weight of the DYKDDDDK-tag (FLAG-tag) is 1012 Da. The multiple polyanionic amino acids in the FLAG tag are less likely to affect the activity of a target protein. The FLAG tag allows highly specific pull-downs that contain low nonspecific background.
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 sequence.
Adding a FLAG-tag to the N- or C-terminus of a protein allows rapid purification of the over-expressed recombinant protein, by using a FLAG-tag specific monoclonal antibody conjugated to agarose beads. Previously, commercial FLAG-tag affinity purification resin is very expensive and can only be used for a couple of times. After many years of development, Sino Biological has developed an excellent FLAG-tag specific monoclonal antibody that can be used for various detection applications as well as be conjugated to agarose for affinity purification. The improved FLAG-tag affinity resin is affordable and can tolerate low pH elution, which increase the life-span of the resin.
In addition, antibodies specific to the FLAG-tag can be used to detect the tagged protein expression level in cell culture, by ELISA and western blot, etc.
FLAG-tagged recombinant protein can be affinity purified directly from a cell culture lysate or supernatant. The FLAG-tagged protein binds to the FLAG-tag specific monoclonal antibody conjugated on an agarose gel. After washing away residual impurities, bound FLAG-tag proteins can be eluted off the affinity column by high concentration of the FLAG-tag peptide or by low pH buffer. For more information on usage of the FLAG-tag affinity purification resin, please refer to: FLAG-tag Affinity Resin.
In some applications, it is desirable to remove the FLAG-tag, for example, for protein crystallization. To allow cleavage of the FLAG-tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the FLAG-tag (FLAG-EK site-protein structure) can allow complete removal of the FLAG-tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the FLAG-tag. For more information on the cleavage site and tag removal by EK and HRV-3C protease, please refer to: Enterokinase (EK), HRV-3C (human rhinovirus protease).
In conclusion, the FLAG tag shows all the advantages and disadvantages of immunoaffinity purification. Although highly selective, the binding capacities are low, making scale-up a costly undertaking.
Young CL, et al. (2012) Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnol J 7(5): 620-634.
Wood DW (2014) New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol 26: 54-61.
DeCaprio J, et al. (2019) Tandem immunoaffinity purification using anti-flag and anti-ha antibodies. Cold Spring Harb Protoc 2019(2).