Hemagglutinin (HA) of influenza virus (epitope tag). HA Peptide is a 9-amino acid synthetic peptide (YPYDVPDYA) that corresponds to a fragment (residues 98-106) of the human influenza virus hemagglutinin (HA) protein. It does not bind to a biological receptor or enzyme; rather, it is a protein tag (epitope tag) recognized by specific anti-HA antibodies. In molecular biology, the HA peptide sequence is genetically fused to a protein of interest (e.g., at the N-terminus or C-terminus), creating an HA-tagged recombinant protein. The HA tag allows for the detection, immunoprecipitation, purification, and cellular localization of the tagged protein using commercially available high-affinity anti-HA antibodies (e.g., monoclonal antibodies 16B12, 12CA5, or polyclonal antibodies). The HA peptide is also used as a competitor to elute HA-tagged proteins from anti-HA antibody resins by adding free HA peptide to disrupt antibody-antigen binding. The HA tag is one of the most commonly used epitope tags in life sciences research. The TFA salt is used to enhance solubility and stability of the synthetic peptide. This product is for research use only and is not a therapeutic agent.

In neutral pH, tagged proteins are frequently isolated from cell culture supernatants and cell lysates using HA Peptide, a highly immunoreactive label. As such, Western blot provides a straightforward method of detection, making them useful instruments for coimmunoprecipitation. The fusion partner protein's biological activity and function are unlikely to be affected by the tiny size of HA Peptide. The HA Peptide is a useful tool for isolating, purifying, detecting, and tracking proteins of interest. It is derived from human influenza hemagglutinin (HA), which corresponds to amino acids 98–106. It is a powerfully immunoreactive epitope. Anti-HA monoclonal antibodies that are highly selective and covalently bound on resin can be used to isolate recombinant HA-tagged proteins. Using TBS containing 1 mg/mL of the HA epitope, HA-tagged proteins can be gently eluted. Three alternatives exist for chemical elution as well: 50 mM NaOH, 3 M NaSCN, and 0.1 M glycine (pH 2-2.8) [1]. For T7 promoter-driven expression in E. coli, even in the absence of transcriptionally active T7 RNAP, the nucleotide sequence encoding the N-terminal HA Peptide in mammalian expression vectors is crucial [2]. According to research findings, caspase 3/7 cleaves the HA peptide, which leaves the immune response completely destroyed. Studies on apoptotic pathways linked to cell death have shown that using HA-tagged proteins and constructs may result in significant artifacts [3].

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In vitro, HA Peptide TFA is used as a competition agent to elute HA-tagged proteins from anti-HA antibody affinity resins. When an HA-tagged protein (e.g., HA-kinase, HA- transcription factor) is bound to an anti-HA antibody resin, the addition of an excess of free HA peptide (0.1-1 mg/mL) competes with the bound HA-tagged protein for the antibody binding site, resulting in the specific elution of the HA-tagged protein. The eluted protein is free of antibody and in its native state, suitable for further functional studies. The peptide is also used as a positive control in Western blotting (dot blot or direct detection). It can be spotted onto a membrane (0.1-10 ug per spot) and detected with an anti-HA antibody to confirm antibody specificity and sensitivity. In immunofluorescence blocking experiments, free HA peptide (10-100 uM) can be pre-incubated with anti-HA antibody to block its binding to HA-tagged proteins, serving as a specificity control. The HA peptide itself is not biologically active; it does not bind to influenza virus or any other receptor. The TFA salt does not affect its use as an epitope competitor. In cell culture, the peptide is not toxic at the concentrations used (0.1-1 mM for short-term incubation). For purification of HA-tagged proteins from cell lysates, the peptide is typically used at a concentration of 0.1-1 mg/mL (approximately 50-500 uM) in elution buffer (e.g., PBS, pH 7.4, or glycine-HCl buffer at pH 3.0 followed by neutralization). The peptide can be custom-synthesized with modifications (e.g., biotinylation, fluorescence labeling) for alternative detection methods.

Not applicable. HA Peptide TFA is not a drug and is not administered to animals for efficacy studies. It is a research reagent used in vitro for protein purification and detection. The peptide may be used in ex vivo assays, such as eluting HA-tagged proteins from tissue lysates, but not for in vivo administration. For a hypothetical in vivo study (not typical), the free peptide (10-100 mg/kg) could be injected intravenously, but it would have no therapeutic effect; it would be rapidly cleared. No such studies are published. The HA Peptide is for laboratory use only.

For non-cellular binding assays, HA Peptide TFA is used in an ELISA or SPR format to measure the affinity of anti-HA antibodies. For an ELISA: coat a 96-well MaxiSorp plate with HA Peptide (0.1-10 ug/mL) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4degC. Block with 3% BSA in PBS for 1 hour at 37degC. Add varying concentrations (0.1-1000 ng/mL) of anti-HA antibody (e.g., 16B12) in PBST (0.05% Tween-20) and incubate for 1-2 hours at room temperature. Wash, add HRP-conjugated secondary antibody (anti-mouse IgG), incubate, and detect with TMB substrate. Measure absorbance at 450 nm. The EC50 of the antibody binding is determined. For a competitive ELISA, incubate a fixed concentration of anti-HA antibody with varying concentrations of free HA Peptide (0.01-1000 uM) for 30 minutes, then add the mixture to the HA Peptide-coated plate. The IC50 of the peptide for blocking antibody binding is determined. For surface plasmon resonance (SPR): immobilize HA Peptide on a CM5 sensor chip via amine coupling. Flow varying concentrations of anti-HA antibody (0.1-1000 nM) over the chip. Calculate the KD (dissociation constant) from association and dissociation curves. The KD for high-affinity anti-HA antibodies is typically in the low nM range (e.g., 1-20 nM). These assays are used to characterize antibodies and to study antibody-antigen interactions. The TFA salt is not used in these assays; the peptide is typically dissolved in PBS or water. The free base is active. For binding to anti-HA resin, a pull-down assay can be performed: Incubate anti-HA antibody resin (e.g., agarose beads) with HA Peptide (0.1-1 mg/mL) in PBS for 1-2 hours at 4degC. Centrifuge, and analyze the supernatant for unbound peptide by HPLC or by BCA assay. The binding capacity of the resin (mg peptide/mL resin) is calculated. This is important for optimizing elution conditions.

For cell-based assays, HA Peptide TFA is not used directly on cells because it is not cell-permeable. However, it is used in protein purification from cell lysates. For example, HEK293 cells expressing an HA-tagged protein are lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors). Clarify the lysate by centrifugation. Add anti-HA antibody resin (e.g., anti-HA agarose beads) to the lysate and incubate for 2-4 hours at 4degC with rotation. Wash the resin 3-5 times with lysis buffer. Elute the HA-tagged protein by adding elution buffer containing HA Peptide TFA (0.1-1 mg/mL in PBS or 50 mM Tris-HCl pH 8.0). Incubate for 30-60 min at 4degC with gentle rocking. Collect the eluate (supernatant). Repeat the elution step once. Pool the eluates and analyze by SDS-PAGE and Coomassie staining or Western blot. The eluted protein can be used for functional assays (kinase activity, DNA binding, etc.). The TFA salt is acceptable; the peptide should be dissolved in elution buffer and the pH adjusted to 7.0-8.0 if necessary. For a control, use a scrambled HA peptide (e.g., YAPDYVPDY) to demonstrate specificity. For immunofluorescence blocking, cells expressing an HA-tagged protein are fixed, permeabilized, and then incubated with anti-HA antibody that has been pre-incubated with HA Peptide (50-200 uM) for 30 minutes. This blocks antibody binding and serves as a specificity control. No staining should be observed in blocked samples. For flow cytometry, a similar blocking experiment can be performed. The peptide is not directly used to treat live cells. All experiments should be performed with appropriate controls. Store HA Peptide TFA as a lyophilized powder at -20degC. Reconstitute in water or PBS at 1-10 mg/mL and store aliquots at -20degC. Avoid repeated freeze-thaw cycles.

Not applicable. HA Peptide TFA is not administered to animals. Therefore, no in vivo animal experimental protocols are available. The peptide may be used in ex vivo assays (e.g., elution of HA-tagged protein from tissue lysates), but it is not injected into animals. For researchers who wish to test the in vivo distribution of the free HA peptide (unlikely), they would label the peptide with a fluorescent dye or radiolabel (e.g., 125I-HA peptide) and inject it intravenously into mice. After 1-4 hours, they would collect organs and measure label content. However, this is not a standard procedure, and no data are published. HA Peptide is a research reagent, not a drug candidate. It is not intended for in vivo use.

No specific pharmacokinetic (PK) data are available for HA Peptide TFA. As a 9-amino acid peptide (MW ~1.1 kDa), if administered intravenously, it would be rapidly cleared from the circulation, with a plasma half-life of minutes (5-15 min) due to glomerular filtration (renal clearance) and proteolytic degradation. It is not orally bioavailable. The TFA salt does not affect PK. The product is not intended for in vivo use; thus, PK parameters are not reported. For research, the peptide is used in vitro only.

No specific toxicity data are available for HA Peptide TFA. As a short peptide composed of naturally occurring L-amino acids, it is generally considered to have low toxicity. In vitro, the peptide (up to 1 mg/mL) is not cytotoxic to HEK293, HeLa, or other cells during short-term (1-2 hour) incubations used for elution protocols. Long-term (24-72 hour) incubation at high concentrations (1-10 mg/mL) may cause some cellular stress due to high peptide load, but this is not typical. The TFA salt (trifluoroacetate) is present in low amounts (equimolar) and is not toxic at the concentrations used. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The product is for research use only and is not intended for human or veterinary use.

[1]. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem. 2013;2013:581093.

[2]. A new idea for simple and rapid monitoring of gene expression: requirement of nucleotide sequences encoding an N-terminal HA tag in the T7 promoter-driven expression in E. coli. Biotechnol Lett. 2012 Oct;34(10):1841-6.

[3]. The HA tag is cleaved and loses immunoreactivity during apoptosis. Nat Methods. 2007 Feb;4(2):107-8.

The HA tag (hemagglutinin tag) is one of the most widely used epitope tags in molecular biology. It is derived from the human influenza virus hemagglutinin protein (residues 98-106, sequence YPYDVPDYA). The HA tag is small (9 amino acids), hydrophilic, and does not interfere with protein folding or function in most cases. It can be fused to the N-terminus, C-terminus, or internally. Anti-HA antibodies (e.g., 12CA5, 16B12, HA-7, HA-11) are commercially available and are used for detection (Western blot, immunofluorescence, immunocytochemistry, flow cytometry), immunoprecipitation (IP), and affinity purification of HA-tagged proteins. The free HA peptide (YPYDVPDYA) is used as a competitor to elute HA-tagged proteins from anti-HA affinity resins under mild, non-denaturing conditions, preserving protein activity. The TFA salt is used to improve solubility and stability of the synthetic peptide. The HA peptide is not a drug and has no therapeutic applications. It is a research-grade chemical for use in laboratories. This product is for research use only and is not approved for clinical or veterinary use.

C55H68F3N9O19

1216.17

HA Peptide;92000-76-5

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :≥ 100 mg/mL (~82.23 mM)
H2O :~50 mg/mL (~41.11 mM)

Solubility in Formulation 1: 1.25 mg/mL (1.03 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (1.03 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (1.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)