PROGEN offers a number of specific primary antibodies recognizing the most frequently used protein tags in cell biological studies including protein-protein interactions, posttranslational modifications, expression dynamics or localization, to laboratory-scale or large-scale production of biotherapeutics.
The use of recombinant proteins for cell biological studies including protein-protein interactions, posttranslational modifications, expression dynamics or localization increased in recent years. To allow rapid identification, modification, production, isolation & purification of proteins/molecules suitable tools including antibodies specifically targeting the protein of interest are needed. A convenient molecular method is the fusion of known protein tags, with high affinity to a specific ligand, to a recombinantly expressed protein.
insertion or improvement of immunoreactive properties
improve solubility
increase expression levels
support protein folding
decrease proteolytic degradation
identification of specific proteins (e.g. WB)
visualization & localization studies (e.g. IF)
isolation & purification (e.g. affinity purification)
analysis of protein-protein interaction
The DDDDK sequence is part of the Flag-tag (patented by Sigma Aldrich).
The intended application of the potential tag is an essential factor and restricts the number of suitable tags to be used. In this regard, it is important to decide either for a small-size or a large-size tag according to the application of interest. Smaller tags usually have less effects on structure, activity or characteristics of the tagged protein. While larger tags can be used to improve features of the target protein. A major concern of using larger tags however is the loss/alteration of the biological activity or increased toxicity of the tagged target protein. It might be necessary to remove large tags by either a specific protease recognition site between the tag and the target protein or sequence encoding for a protein with self-splicing capacities.
Another consideration is the location of the tag within the protein of interest. Most of the tags are preferrably placed at the C- or N-terminus of the target protein to avoid interference with active sites. The positioning of the tag at one of the termini moreover ensures the exposure of the tag to the surface of the protein in order to allow the interaction with its ligand (e.g. for purification). In general, the oligonucleotide encoding for a tag is prevalently inserted at the 5’ end of the gene of interest to ensure a good translational initiation. In some cases it might also be possible to choose a location inside the gene sequence.
In order to increase the sensitivity of a tag, especially small-size tags, it is possible to attach tandem copies increasing signal strength and reinforcing signal-to-noise ratio. It is also possible to use a tandem tag system combining features of different tag groups. This is mainly used in the Tandem-Affinity Purification (TAP) allowing the isolation of protein complexes and the reduction of non-specific background based on two consecutive purification steps.
There are two standard methods for the attachment of a tag to your protein of interest. First, genomic tagging by the introduction of an oligonucleotide encoding for the respective tag into the genomic coding sequence of the target protein. Second, the more prevalent way, by the introduction of the target protein coding sequence into an expression vector containing the desired tag sequence. The majority of modern expression vectors for mammalian, yeast, insect or E.coli cells carry one or even multiple tags. The vector-based method comes with several advantages, e.g. it is less labor-intense and much easier compared to the genomic tagging. Furthermore, this method allows the discrimination between the wild-type protein (untagged) and the tagged protein in a cell for analysis.