FLAG tag Peptide (DYKDDDDK): Atomic Benchmarks for Recombinant Protein Purification

Executive Summary: The FLAG tag Peptide (DYKDDDDK) is an 8-amino acid synthetic peptide widely used as an epitope tag for recombinant protein purification and detection. The peptide is highly soluble, with solubility exceeding 210.6 mg/mL in water and 50.65 mg/mL in DMSO at room temperature. It features an enterokinase-cleavage site, enabling gentle and specific elution of FLAG fusion proteins from anti-FLAG M1 and M2 affinity resins. Verified purity exceeds 96.9% by HPLC and mass spectrometry. The peptide is not suitable for eluting 3X FLAG fusion proteins; for those, a 3X FLAG peptide is required. These properties are supported by peer-reviewed structural biology and biochemical benchmarks (Ghanbarpour et al., 2025).

Biological Rationale

The FLAG tag Peptide (DYKDDDDK) is engineered as an epitope tag for recombinant protein expression, detection, and purification. Its sequence, DYKDDDDK, is recognized by high-affinity monoclonal antibodies (anti-FLAG M1 and M2), enabling selective capture and elution of tagged proteins (product page). The peptide includes an enterokinase cleavage site (DDDDK), allowing specific enzymatic removal post-purification. The tag's small size minimizes interference with protein folding and function (Annexin-V-FITC.com). It is widely used in prokaryotic and eukaryotic systems to facilitate proteomics, interactome mapping, and structural biology workflows. By enabling gentle elution, the FLAG tag Peptide preserves protein integrity, supporting downstream enzymatic and functional assays.

Mechanism of Action of FLAG tag Peptide (DYKDDDDK)

The FLAG tag Peptide functions as a protein purification tag peptide by providing a specific epitope for antibody-based affinity capture. The DYKDDDDK sequence binds with nanomolar affinity to anti-FLAG M1 or M2 monoclonal antibodies immobilized on chromatography resins. Gentle elution is achieved via competitive displacement when excess free FLAG tag Peptide is added to the column, or by enzymatic cleavage at the enterokinase site. This mechanism allows isolation of recombinant proteins under non-denaturing conditions, preserving native structure and activity. The minimal size of the tag reduces steric hindrance and the risk of altering the biochemistry of the target protein (BuyBrivanib.com). The peptide does not efficiently displace 3X FLAG-tagged proteins, as these require a higher-affinity 3X FLAG peptide for elution.

Evidence & Benchmarks

• FLAG tag Peptide (DYKDDDDK) exhibits solubility >210.6 mg/mL in water, >50.65 mg/mL in DMSO, and >34.03 mg/mL in ethanol at room temperature (product page).
• Purity is confirmed at >96.9% by HPLC and mass spectrometry (A6002, ApexBio).
• The DYKDDDDK sequence provides a high-affinity epitope for anti-FLAG M1 and M2 antibodies, enabling selective purification of tagged proteins (Ghanbarpour et al. 2025, https://doi.org/10.1038/s44318-025-00408-1).
• The enterokinase-cleavage site permits site-specific removal of the FLAG tag after purification (Ghanbarpour et al. 2025, DOI).
• Elution of FLAG-tagged proteins from anti-FLAG resins is achieved at a typical concentration of 100 μg/mL peptide in buffer (A6002, ApexBio).
• FLAG tag DNA sequence: 5'-GACTACAAAGACGACGACGACAAG-3'; Nucleotide sequence: 5'-GAC TAC AAA GAC GAC GAC GAC AAG-3' (see DMG-PEG2000.com).
• Storage at -20°C, desiccated, is recommended for peptide stability; solutions should be prepared fresh and used promptly (ApexBio).
• FLAG tag Peptide does not efficiently elute 3X FLAG fusion proteins; alternative reagents are required (Flag-Peptide.com).

Applications, Limits & Misconceptions

The FLAG tag Peptide is widely used in recombinant protein purification, detection, and interactome studies. It is compatible with a range of affinity resins and detection assays, including Western blotting, ELISA, and immunoprecipitation. Its high solubility and gentle elution make it suitable for sensitive proteins and downstream enzymatic assays. However, the peptide is not designed to elute 3X FLAG-tagged proteins or for use in systems where antibody cross-reactivity or protease contamination may occur. Use of the FLAG tag Peptide (DYKDDDDK) extends the findings of Annexin-V-FITC.com by providing updated benchmarks for solubility and elution conditions, and contrasts with BuyBrivanib.com by clarifying the limitations for 3X FLAG systems.

Common Pitfalls or Misconceptions

• FLAG tag Peptide (DYKDDDDK) does not efficiently elute proteins tagged with 3X FLAG; a 3X FLAG peptide is required for those fusion proteins (Flag-Peptide.com).
• Long-term storage of peptide solutions is discouraged; freshly prepared solutions ensure maximal activity (ApexBio).
• Antibody cross-reactivity may occur in some systems; validation with negative controls is necessary (Phenyl-Sulfate.com).
• Protease contamination can degrade the tag or fusion protein; use protease inhibitors when necessary.
• The tag may be inaccessible if fused to a buried region of the target protein; optimal fusion design is required.

Workflow Integration & Parameters

The FLAG tag Peptide (DYKDDDDK) integrates into recombinant protein workflows at the cloning, expression, and purification stages. Fusion of the DYKDDDDK sequence to the N- or C-terminus of the target protein is achieved via standard molecular cloning, using the DNA sequence 5'-GACTACAAAGACGACGACGACAAG-3'. Expression is carried out in prokaryotic or eukaryotic systems as needed. Lysis is performed under non-denaturing conditions to preserve tag accessibility. Affinity capture uses anti-FLAG M1 or M2 resins, followed by gentle elution with 100 μg/mL FLAG tag Peptide in buffer, or enzymatic release using enterokinase. For high-throughput or automated workflows, the peptide's high solubility allows for rapid preparation of elution buffers. Storage of the solid peptide is at -20°C, desiccated, with minimal freeze-thaw cycles. For detailed integration strategies, see DMG-PEG2000.com, which we extend here by providing direct, quantitative solubility data and updated elution protocols.

Conclusion & Outlook

The FLAG tag Peptide (DYKDDDDK) is a validated epitope tag providing high specificity, robust solubility, and gentle elution for recombinant protein workflows. Atomic, quantitative benchmarks support its widespread adoption in structural biology and proteomics. Its limitations, including incompatibility with 3X FLAG systems and the need for fresh solutions, are well defined. Ongoing advances in antibody engineering and tag design may further expand its utility. For ordering and further details, see the A6002 kit product page.