FLAG Tag: Definition, Overview, & Applications

FLAG Tag: Definition, Overview, & Applications

FLAG TAG: Overview

The FLAG tag is a protein tag widely used in protein purification procedures for the high specificity and purity of target proteins. When high purity is required, it is commonly used as an alternative or follow-on to His tags.

The tag is also known as DYKDDDDK-tag (D=Aspartic acid; K=Lysine; Y=Tyrosine), based on the amino acid sequence of the tag, FLAG octapeptide, or FLAG epitope.

The tag is of 1012.9 Daltons, or roughly 1 kDa, and added either to the C-terminal or N-terminal of a protein. Moreover, the tag is also used in tandem formation, which means multiple tags are added adjacent to each other.

It can be in the form of (-DYKDDDDK-DYKDDDDK-DYKDDDDK). Moreover, in some applications, a modified or altered FLAG tag is also used, such as  DYKDHDG-DYKDHDI-DYKDDDDK.

The FLAG tag is one of the most specific protein tags used in affinity purification or protein expression studies. However, some alternatives to the tags are also available, such as the Rho1D4 epitope tag and CL7 tag.

Below is a chart with the chemical characteristics of the FLAG tag.

S.No.FeaturesDescription1Amino acid sequenceDYKDDDDK2Size1012.9 dalton ~ 1 kDa3Specificity of interaction (KD)– corresponding to the antibody100 nM (3)4Tandem useCan be used in tandem or as or as 3xFLAG5Affinity tag useCan be used in combination with affinity tags6Binding to magnetic bead or purification resinBinds via DYKDDDDK Flag tag antibody (approx. 150 kDa)7Affinity matrixAnti-FLAG Antibody (such as anti-DYKDDDDK)

In this article, we will review the working mechanism of the FLAG tag (how it acts when used in a molecular process) and more about their lab applications.

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How Does FLAG Tag Work?

FLAG tags are added to proteins at N-terminus or C-terminus using recombinant DNA technology. The terminus best suited for a particular protein needs to be tested individually for each application.

The peptide sequence of the FLAG-tag can either be singly added to the protein or in tandem with a 3xFLAG peptide: DYKDHD-G-DYKDHD-I-DYKDDDDK. Such a sequence contains a final tag as enterokinase (a protease enzyme that cleaves acidic peptides) cleavage site.

The FLAG-tagged protein identifies and binds to the FLAG tag-specific monoclonal antibody conjugated on a magnetic bead or agarose gel. Then, residual impurities are washed away, and the FLAG-tagged proteins are eluted using an affinity column with a low pH buffer or a high concentration of FLAG tag peptide.

After purification is complete, the enterokinase enzyme cleaves the FLAG tag at the recognition site Asp-Asp-Asp-Asp-Lys-X (where X is any amino acid) present at the C-terminus of the amino acid lysine, leaving only the target proteins.

Besides being used alone in the applications, FLAG tags can also be used in conjunction with the other epitope tags, such as HA-tag, His-tag, GST-tag, or myc-tag.

Moreover, the antibodies used in the purification stem are often position-sensitive and only recognize the tag at N-terminus. However, some, like M2 antibodies, are position-insensitive and can identify the TAG present anywhere in the protein sequence.

Advantages of Using FLAG Tag

The advantages of using FLAG tags are:

  • When used as a fusion protein, FLAG tags do not react with the target protein, which can affect its physical and chemical proteins and metabolic functions.
  • FLAG is easily recognized by antibodies, which makes it convenient to use it for lab applications like western blotting and ELISA.
  • FLAG tag fused with the target protein is used in applications like affinity chromatography to achieve high purification efficiency.

What is Flag Tag Used For?

The FLAG tag can be used in applications requiring antibodies for protein recognition. In cases where no antibodies are involved, the tags are conjugated to a protein (forming fusion protein) to perform the lab applications.

The FLAG tag has major application in protein purification from mammalian expression systems or cell lines and in studies like immunoprecipitation, immunofluorescence (for protein localization), ELISA, immunostaining, SDS-PAGE protein electrophoresis (for protein detection), flow cytometry, or western blotting assays.

Protein Purification

FLAG tags are used to purify proteins from cell culture lysates or supernatants. The steps involved in the workflow are:

  • The FLAG tags are attached to the N-terminus or C-terminus of the target proteins.
  • The tagged protein binds to the specific monoclonal antibodies conjugated on the matrix of magnetic beads or agarose gel.
  • The residual impurities are washed off using the reagent washing buffer.
  • Then, the bound proteins are eluted off the affinity column using a low pH buffer or a high concentration of the FLAG tag peptide.


FLAG tags have applications in a range of assays involving antibodies for identification, such as immunoprecipitation, immunofluorescence, and ELISA. These assays either involve a single conjugated primary antibody or a primary antibody with a specific conjugated secondary antibody for the protein expression or localization studies.

Some other assays like SDS-PAGE electrophoresis and western blotting assays involve the use of FLAG tags for protein detection.

What Industries Use FLAG Tag?

FLAG tags have massive applications in life sciences labs and industries and pharmaceutical sectors to perform workflows like western blotting, immunoprecipitation, and protein purification assays for several purposes.


FLAG peptide is used as an artificial antigen to develop high-affinity monoclonal antibodies for applications like affinity chromatography for protein purification from a variety of cell lysates or protein localization within living cells.


In the pharma industry, FLAG tags are used to separate overexpressed or recombinant proteins from wild-type proteins. Moreover, it also has applications in the isolation of protein complexes with multiple protein subunits.

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FLAG tags are epitope proteins with DYKDDDDK sequence, where D=Aspartic acid; K=Lysine; Y=Tyrosine. It’s a highly specific protein used for assays that involve antibodies for identification, like protein purification and immunological assays, such as immunoprecipitation and immunofluorescence.

In the process, the FLAG tag is attached to the protein, which is subjected to a matrix having anti-FLAG antibodies as conjugates. The tag binds to the antibody, and the protein is eluted using a suitable solution or pH level.

Because the FLAG tag does not react with the chemical in workflows and offers high efficiency in protein studies, it’s widely used in industries and pharmaceutical industries. Some other alternatives of the tag like myc-tag and RhoD tags are also available to suit a particular workflow.

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