Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Tagging with xxxDDDDK may be done at the N-terminus, N-terminus preceded by a methionine residue, C-terminus, and in internal positions of the target protein. The small size of the epitope tag and its high hydrophilicity tend to decrease the possibility of interference with protein expression, proteolytic maturation, antigenicity and function. The enterokinase cleavage site allows it to be completely removed from the purified fusion proteins.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Prior to conjugation the antibody was analyzed by western blot using various amino-terminal and carboxy-terminal DDDDK fusion proteins and Invitrogen Positope™ R900-40. A 1/5000 dilution gave bands against 0.1, 1.0 and 10 ng of the fusion proteins and 100 ng and 25 ng of the Positope.
Optimal working dilutions should be determined for your particular assay conditions.
This is a useful tool for the localisation and characterisation of DDDDK tagged proteins.