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Short answer: A FLAG tag is a synthetic eight-amino-acid epitope tag with the sequence DYKDDDDK, fused to the N- or C-terminus of a recombinant protein so that an anti-FLAG antibody can detect or capture it. Its DNA sequence is commonly given as GAC TAC AAG GAC GAC GAT GAC AAG. The 3xFLAG tag is longer (22 amino acids) and is not a simple threefold repeat of the 1x tag.
This page is about the molecular-biology tag. It is unrelated to flag-tag games, flag football or any other use of the phrase; the tag is the DYKDDDDK epitope used on proteins in cell lysate and purified samples.
What is the sequence of a FLAG tag?
The 1x FLAG tag is the octapeptide DYKDDDDK (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Proteintech’s technical notes give its theoretical molecular weight as 1012.98 Da and its theoretical pI as 3.97, and list the coding sequence as GAC TAC AAG GAC GAC GAT GAC AAG (24 nucleotides). Excedr’s overview also calls it the FLAG octapeptide or epitope, at roughly 1,013 Da, and notes it can be placed at the N- or C-terminus; the best position is tested per application.
The DNA string above is one codon choice for the peptide. Because the genetic code is degenerate, a vector can encode DYKDDDDK with different codons, so when you need the exact nucleotides, read them from the sequence map of the vector you are actually using.
FLAG tag vs 3xFLAG tag: what are the differences?
The 3xFLAG tag is a 22-amino-acid sequence, DYKDHD-G-DYKDHD-I-DYKDDDDK, which Proteintech describes as not a simple repeat of the 1x tag. Its theoretical molecular weight is 2730.71 Da (pI 4.16), about 2.7 times the mass of the 1x tag, and it carries more charged residues (11 negative, 4 positive).
- Affinity: 3xFLAG gives higher antibody affinity, so it is often preferred for demanding or tandem purification.
- Size: 1x FLAG is smaller and less likely to interfere with the fusion partner, and is described as sufficient for Western blot detection.
- Elution trap: a 1x FLAG peptide will not elute a 3xFLAG-tagged protein, so the competing peptide has to match the tag format on the protein.
What is His-tag sequence?
The His-tag (polyhistidine tag, 6xHis) is a string of histidine residues, described by Thermo Fisher as six to nine histidines, fused to the N- or C-terminus of a recombinant protein. A six-histidine tag is written HHHHHH. It binds metal ions on a nickel-NTA resin, and Thermo’s guidance uses a binding and wash buffer containing 10-25 mM imidazole to limit non-specific binding, and a high imidazole concentration (at least 200 mM) to elute. For a fuller purification workflow see protein purification and affinity tags.
How the tags are used in practice
Detection and capture with anti-FLAG antibodies
Excedr reports that anti-FLAG antibodies are often position-sensitive, recognising the tag only at the N-terminus of the protein, while the M2 antibody is position-insensitive. That is why the terminus you tag decides which antibody or resin can be used. For lysis conditions and controls in capture experiments, see co-immunoprecipitation controls and interpretation, and for the detection step see Western blot protocol basics.
Elution and tag removal
Proteintech lists three ways to release FLAG-tagged protein: competitive elution with the 1x or 3xFLAG peptide, glycine at pH 2.5, or SDS sample buffer. The DDDDK stretch is an enterokinase recognition site, so the tag can be cleaved off; Excedr gives the cleavage site as Asp-Asp-Asp-Asp-Lys-X.
Checklist: FLAG, 3xFLAG and His-tag side by side
| Feature | 1x FLAG | 3xFLAG | His-tag (6xHis) |
|---|---|---|---|
| Sequence | DYKDDDDK (8 aa) | DYKDHD-G-DYKDHD-I-DYKDDDDK (22 aa) | Six to nine histidines |
| Theoretical mass | 1012.98 Da | 2730.71 Da | Varies with tag length |
| Position | N- or C-terminus | N- or C-terminus | N- or C-terminus |
| Capture and release | Anti-FLAG antibody; peptide, pH 2.5 glycine or SDS buffer | Anti-FLAG antibody; needs the 3xFLAG peptide, not the 1x peptide | Metal (Ni-NTA) resin; at least 200 mM imidazole |
Figures in the columns come from Proteintech (FLAG and 3xFLAG) and Thermo Fisher (His-tag).
Matching the tag to your reagents
When you plan reagents for an expression project, check three things against the construct design: the tag format (1x or 3x) against the elution peptide, the tag position (N- or C-terminus) against the antibody’s position sensitivity, and the tag type against the resin chemistry (antibody resin for FLAG, metal resin for His). Cloning and expression background is in gene cloning and protein expression in E. coli.
Common misunderstandings
- 3xFLAG is not three copies of the same eight residues. Two of its three units read DYKDHD and are joined by single-residue linkers, so it cannot be built by pasting the 1x sequence three times.
- A FLAG tag is not a natural protein. It is an artificial peptide used for detection and purification.
- One elution reagent does not fit both formats. A 1x peptide will not release a 3xFLAG fusion.
FAQ
What are the differences between FLAG tag and 3x-Flag-tag?
1x FLAG is DYKDDDDK (8 aa, 1012.98 Da); 3xFLAG is 22 aa (2730.71 Da), binds antibody more tightly, and requires its own peptide for competitive elution.
What is the sequence of a FLAG tag?
DYKDDDDK at protein level; GAC TAC AAG GAC GAC GAT GAC AAG is the commonly listed DNA sequence.
What is His-tag sequence?
A run of histidines, typically six (HHHHHH) and up to nine, at the N- or C-terminus.
Sources
- Proteintech Group, FLAG tag and 3x FLAG tag, checked 2026-10-29.
- Excedr, FLAG tag overview, checked 2026-10-29.
- Thermo Fisher Scientific, His-tagged proteins: production and purification, checked 2026-10-29.
