Influenza Hemagglutinin (HA) Peptide: Precision Epitope Tag for Protein Detection and Purification

Executive Summary: The Influenza Hemagglutinin (HA) Peptide (sequence: YPYDVPDYA) is a synthetic nine-amino acid tag derived from the influenza virus hemagglutinin protein.^[product] It is used as a high-purity (>98%) molecular tag for fusion protein detection and purification.^[1] The HA tag facilitates immunoprecipitation by competitively binding to anti-HA antibodies, enabling precise elution of target proteins.^[internal] Its high solubility across DMSO, ethanol, and water ensures compatibility with diverse workflows.^[product] Proper storage and handling are essential for optimal stability and performance.

Biological Rationale

The HA tag is derived from the epitope region of the influenza virus hemagglutinin (HA) surface glycoprotein. This nine-amino acid sequence (YPYDVPDYA) is recognized with high specificity by monoclonal anti-HA antibodies.^[1] The minimal size of the peptide reduces steric hindrance, making it suitable for fusion to proteins without altering their function or localization. Such tags are broadly applied to track protein expression, facilitate detection, and mediate purification in molecular and cell biology research. In particular, the HA tag has enabled detailed studies of protein-protein interactions, post-translational modifications, and vesicular trafficking.^[internal] The sequence is highly conserved and does not naturally occur in most eukaryotic or prokaryotic systems, minimizing background signal.^[product]

Mechanism of Action of Influenza Hemagglutinin (HA) Peptide

The Influenza Hemagglutinin (HA) Peptide acts as a competitive ligand for anti-HA antibodies. When HA-tagged fusion proteins are incubated with immobilized anti-HA antibodies (e.g., on magnetic beads or agarose), the specific antibody-epitope interaction enables selective capture of the target protein. To elute the bound fusion protein, a molar excess of free HA peptide is introduced, which competitively displaces the HA-tagged protein from the antibody via reversible binding.^[internal] This mechanism preserves the structural integrity and post-translational modifications of the target protein, as elution does not require harsh denaturants or changes in pH. The high affinity and specificity of the interaction make the HA peptide suitable for use in immunoprecipitation, co-immunoprecipitation, and affinity purification protocols.^[product]

Evidence & Benchmarks

• The HA tag (YPYDVPDYA) is specifically recognized by monoclonal antibodies (clone 12CA5), allowing detection at sub-nanogram levels in Western blot and ELISA assays (Wei et al., 2021).
• HA peptide exhibits solubility ≥55.1 mg/mL in DMSO, ≥100.4 mg/mL in ethanol, and ≥46.2 mg/mL in water at 20°C, supporting use in diverse buffers and conditions (ApexBio, A6004).
• Immunoprecipitation using anti-HA magnetic beads and competitive elution with free HA peptide preserves native protein complexes for downstream analysis (internal review).
• Purity of commercial HA peptide (A6004) is confirmed to be >98% by HPLC and mass spectrometry (ApexBio).
• Storage at -20°C in desiccated conditions ensures peptide stability for at least 12 months; reconstituted solutions should not be stored long-term (ApexBio).

Applications, Limits & Misconceptions

The Influenza Hemagglutinin (HA) Peptide is routinely used for the following applications:

• Epitope tagging of recombinant proteins for detection and localization studies in cellular and animal models.
• Affinity purification of HA-tagged proteins via immunoprecipitation and subsequent competitive elution with free HA peptide.
• Protein-protein interaction assays, including co-immunoprecipitation, to dissect molecular complexes in mechanistic studies (internal link).
• Post-translational modification research, such as ubiquitination and SUMOylation pathway analysis (internal link).

Common Pitfalls or Misconceptions

• The HA peptide does not provide cell membrane permeability; it cannot deliver proteins into living cells.
• HA tag detection is dependent on the availability of high-affinity monoclonal anti-HA antibodies; polyclonal antibodies may yield nonspecific results.
• The peptide does not function as a protease cleavage site and should not be used for proteolytic removal of tags.
• Overuse of HA peptide during elution can lead to antibody saturation, reducing the efficiency of subsequent runs.
• HA tag fusion may interfere with protein function if the tag is inserted into a critical structural domain; empirical validation is needed for each construct.

Compared to previous reviews, this article provides updated solubility, purity, and storage parameters for the A6004 kit, clarifying optimal use cases and addressing recent advances in competitive elution strategies.

Workflow Integration & Parameters

• Tagging: Clone the HA tag (YPYDVPDYA) in-frame at the N- or C-terminus of the target protein using standard molecular biology techniques. Ensure the reading frame and sequence context are preserved.
• Expression: Express the HA-tagged fusion protein in the desired system (e.g., mammalian, yeast, or bacterial cells). Confirm expression by Western blot using anti-HA antibodies.
• Capture: Incubate lysates with anti-HA magnetic beads or agarose under native or denaturing conditions, depending on the target protein's solubility and stability.
• Elution: Add free HA peptide (recommended 1–3 mM final concentration), in buffer compatible with downstream applications, to competitively elute the HA-tagged protein.
• Storage: Store lyophilized HA peptide desiccated at -20°C. Avoid repeated freeze-thaw cycles. Use freshly prepared solutions; do not store working solutions long-term.

For detailed integration in advanced protein-protein interaction and ubiquitination workflows, see the article "Redefining Protein Interaction and Ubiquitination Research", which this review extends with updated benchmarks and protocol clarifications.

Conclusion & Outlook

The Influenza Hemagglutinin (HA) Peptide remains a gold-standard molecular tag for protein detection and purification, offering high specificity, solubility, and adaptability across experimental platforms. Recent improvements in peptide purity, storage guidance, and competitive elution protocols enhance its reliability in mechanistic and translational research. As protein interaction and post-translational modification studies advance, the A6004 HA peptide provides a validated, versatile foundation for reproducible molecular biology workflows. For product details and ordering, see the ApexBio Influenza Hemagglutinin (HA) Peptide page.