FLAG® System

The FLAG® Expression System is a proven method to express, purify and detect recombinant fusion proteins containing the DYKDDDDK epitope.

Overview

The FLAG Expression System utilizes a short, hydrophilic 8-amino acid peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys or DYKDDDDK) which is fused to the recombinant protein of interest when expressed from a pFLAG vector. These small hydrophilic tags significantly improve detection and purification of recombinant fusion proteins when combined with the highly specific and sensitive ANTI-FLAG antibodies. In addition, because of the small size of the FLAG® peptide tag, it is not likely to obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein.

Sigma′s unique, highly specific monoclonal (M1, M2, M5) and polyclonal anti-FLAG antibodies, each contain unique recognition and binding characteristics to the FLAG peptide sequence (Fig.1). For purification and immunoprecipitation, the highly specific FLAG M2 is conjugated onto magnetic and agarose affinity resins. The bound recombinant protein is subsequently eluted using the correct FLAG peptide.

Sensitivity of 3X FLAG

3X FLAG is the ideal epitope when evaluating low-level protein expression. The 3X FLAG system improves upon the original system by fusing 3 tandem FLAG epitopes to a recombinant protein (Fig.1). The 3X FLAG system has been found to be 20-200 times more sensitive than any other system (Fig.2) with detection limits below 10 femtomoles.

Vectors for Bacterial, Insect and Mammalian Systems

Bacterial FLAG® expression vectors offer a choice of periplasmic or cytoplasmic expression. All of the E. coli expression vectors and Insect Expression Vectors confer ampicillin resistance for easy selection of positive transformants.
Mammalian FLAG expression vectors offer a choice of transient or stable transfection, for either cytoplasmic or extracellular expression.
Confidently express your FLAG or 3X FLAG sequence on the N- or C-terminal, knowing there is an enterokinase (Ek) cleavage site to remove the fusion tag if necessary.
Applications

FLAG and 3xFLAG products have been used in protein purification, Western blotting, immunocytochemistry, immunoprecipitation, immunofluorescence, ELISA, electron microscopy, flow cytometry, supershift assays, in the study of protein-protein interactions, cell ultrastructure, and protein localization. Browse additional application references or submit your own in our FLAG® Literature portal.

Figure 1. The FLAG and 3X FLAG Amino Acid Sequences. The 3X FLAG system is an improv
Figure 2. Comparative Sensitivity of 3X FLAG. Each protein fusion tag was cloned sep
An Overview of the FLAG Protein Expression System