Recombinant Protein Purification & Detection

FLAG® System

Insist on the Original & Proven Performer

The FLAG Expression System is a proven method to express, purify and detect recombinant fusion proteins. FLAG and 3xFLAG are useful in Western blotting, immunocytochemistry, immunoprecipitation, flow cytometry, protein purification, and in the study of protein-protein interactions, cell ultrastructure, and protein localization. These small hydrophilic tags significantly improve detection and purification of recombinant fusion proteins when used with our highly specific and sensitive ANTI-FLAG® antibodies.

  • Sequence is highly charged and useful for sensitive detection
  • Sensitivity can be enhanced using the 3x FLAG epitope
  • Facilitates the study of low-abundance proteins and the optimisation of difficult protein expression projects

Overview of FLAG-Protein Expression System
Vectors for Bacterial and Mammalian Systems

Comparative Sensitivity of 3xFLAG
Cytochemical Staining using FLAG

FLAG and 3xFLAG Products

 Overview of FLAG-Protein Expression System

The FLAG system utilizes a short, hydrophilic 8-amino acid peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) which is fused to the recombinant protein of interest when expressed from a pFLAG vector. The FLAG peptide includes the binding site for several highly specific ANTI-FLAG monoclonal antibodies (M1, M2, M5) and polyclonal antibodies and conjugates, each with different recognition and binding characteristics. The FLAG peptide is likely to be located on the surface of a fusion protein because of its hydrophilic nature. As a result, the FLAG peptide is more likely to be accessible to antibodies and to cleavage by enterokinase (Ek). 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.

The 3X FLAG system improves upon the original system by fusing 3 tandem FLAG epitopes to a recombinant protein (Fig. 1). A 10-20 fold increased detection enhancement has been shown using 3X FLAG fusions (Fig. 2, 3). As with the original FLAG tag, it is hydrophilic, contains an enterokinase cleavage site and is relatively small (22 amino acids), therefore, the risk of altering protein function, blocking other epitopes or decreasing the solubility is minimized.

View an overview of the FLAG Protein Expression System

FLAG and 3XFLAG Amino Acid Sequence


 Vectors for Bacterial and Mammalian Systems

  • Cytoplasmic expression or secretion
  • Bacterial vectors with T7 or tac promoters
  • 3XFLAG, as well as FLAG, offered for mammalian systems
  • Options for dual-tagged fusion proteins
  • N-terminal FLAG and 3×FLAG vectors provide an Ek cleavage site for removal of the fusion tag
  • New BICEP™ line of vectors for bicistronic expression in mammalian cells; offer high performance stable expression of FLAG fusions or multi-gene expression possibilities

Purification FLAG Vector


 Comparative Sensitivity of 3X FLAG

  • Ultrasensitive; 20–200 times more sensitive than any other system
  • Detect <10 femtomoles
  • Ideal for cases of low-level expression in mammalian cells
  • 3 tandem repeats of the FLAG sequence
  • Enhanced detection for immunoprecipitation, Western blots, and immuno cytochemistry

Comparative Sensitivity of 3X FLAG®. Each protein fusion tag was cloned separately on the C-terminal of GST.

Each protein fusion tag was cloned separately on the C-terminal of GST.


 Cytochemical Staining using FLAG

Cytochemical Staining using FLAG

FLAG-p53 and mock transfected COS-7 cells were permeabilized, fixed, blocked, and incubated with Anti-FLAG M2, affinity purified antibody.