HA-tag Peptide PAT5E7AT Antibody

What is the HA-tag and why is it frequently used in protein research?

The HA (hemagglutinin) tag is a nine amino acid peptide sequence (YPYDVPDYA) derived from the human influenza virus hemagglutinin protein. It has become widely adopted in molecular biology research due to several advantageous properties. The tag is relatively small, minimizing interference with protein structure and function while providing a reliable epitope for antibody recognition. Researchers frequently employ HA tags because they enable protein detection and purification without requiring protein-specific antibodies, which can be particularly valuable for newly discovered or poorly immunogenic proteins . Additionally, the HA tag has been extensively characterized and validated across multiple experimental systems, making it a dependable choice for protein tracking and analysis .

What are the key specifications of the HA-tag Peptide PAT5E7AT Antibody?

The HA-tag Peptide PAT5E7AT Antibody (catalogue number ANT-730) is a Mouse Anti-Human Monoclonal antibody derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with a synthetic peptide. It belongs to the Mouse IgG1 subclass with kappa light chain. The antibody is supplied as a sterile filtered colorless solution at a concentration of 1mg/ml in PBS (pH 7.4) containing 10% glycerol and 0.02% sodium azide. It has been purified from mouse ascitic fluids using protein-A affinity chromatography and has demonstrated specificity and reactivity in ELISA and Western blot analyses . This particular clone has been optimized for research applications requiring high specificity for the HA epitope tag.

What are the primary applications for HA-tag Peptide PAT5E7AT Antibody in research settings?

The HA-tag Peptide PAT5E7AT Antibody has been validated for several critical research applications. It performs effectively in Western blot analysis for detecting HA-tagged proteins expressed in cell lysates. The antibody has also been tested for immunoprecipitation assays, allowing researchers to isolate HA-tagged protein complexes from cellular extracts . Additionally, it can be used in immunocytochemistry/immunofluorescence analysis to visualize the subcellular localization of HA-tagged proteins within fixed cells . Some researchers have also employed this antibody in ELISA applications for quantitative detection of tagged proteins. Each application requires specific optimization and titration to achieve optimal results based on the particular experimental conditions and the protein being studied.

What are the quantitative differences in signal generation between primary and secondary antibodies in HA-tag detection systems?

Quantitative analysis of HA-tag detection systems reveals important considerations regarding antibody limiting factors. Research has demonstrated that in many experimental setups, the secondary antibody—not the primary anti-HA antibody—represents the limiting reagent in signal generation. Comparative studies found that diluting the secondary antibody resulted in a proportional decrease in signal intensity, while a 1:100 dilution of primary anti-HA antibody caused only a 60% reduction in specific signal . This suggests that typical protocols often use primary anti-HA antibodies at concentrations far exceeding those necessary for effective epitope binding.

These findings have significant implications for experimental design and resource allocation. Researchers working with the PAT5E7AT antibody should consider optimizing secondary antibody concentrations while potentially reducing primary antibody usage for cost-effective signal generation without compromising detection sensitivity .

How can researchers effectively normalize HA-tag signal intensity when comparing different HA-tagged proteins?

Normalizing HA-tag signal intensity presents a significant challenge when comparing different HA-tagged proteins, particularly in quantitative analyses. A robust normalization strategy involves implementing a competition assay where the in vitro translated peptides or proteins are designed to include a single HA tag, theoretically interacting with the anti-HA antibody with equal affinity regardless of the sequence N-terminal to the HA tag . This approach enables quantification independent of the specific protein sequence.

For more precise normalization in comparative studies, researchers should consider:

• Implementing parallel translation efficiency controls using radiolabeled amino acids to quantify relative translation rates

• Utilizing acoustic membrane microparticle technology (AMMP) for target-binding assays where immobilized target proteins on magnetic beads bind to HA-tagged synthetic peptides

• Employing sandwich assay configurations where fluoresceinated anti-HA antibodies bind to HA-tagged peptides as well as to sensor-immobilized antibodies

This methodological approach increases quantitative precision by 4- to 10-fold compared to traditional methods and enables accurate rank-ordering of different HA-tagged proteins or peptides based on relative binding affinities rather than expression differences .

What are the comparative performance metrics between PAT5E7AT and other anti-HA antibody clones in detecting low-abundance proteins?

Quantitative comparison of different anti-HA antibody clones reveals significant performance variations when detecting low-abundance proteins. While the PAT5E7AT clone demonstrates reliable detection capabilities, comparative studies suggest that the AF291 antibody clone may offer optimal sensitivity for HA epitope detection in limiting conditions . When antibody concentrations were reduced to 50 ng·mL−1, AF291 maintained stronger signal generation compared to several other clones.

It's worth noting that while recently developed antibodies against rationally designed epitopes are sometimes presumed superior, comparative data does not consistently support this assumption. For researchers working with low-abundance proteins, selection of the appropriate anti-HA antibody clone should be based on empirical testing within their specific experimental system rather than general claims .

How should researchers optimize western blot protocols for maximum sensitivity with HA-tag Peptide PAT5E7AT Antibody?

Optimizing western blot protocols for the HA-tag Peptide PAT5E7AT Antibody requires attention to several critical parameters. Based on experimental data, researchers should consider the following methodological approach:

• Sample preparation: Load adequate protein quantities (30-90 μg of whole cell lysate for standard applications) to ensure detection of HA-tagged proteins, particularly for low expression systems .

• Transfer optimization: Use nitrocellulose membranes for optimal antibody binding and protein retention. Evidence suggests that PVDF membranes may also be effective, particularly when working with hydrophobic tagged proteins .

• Blocking conditions: Implement 2-5% BSA or non-fat dry milk in TBS-T for 1 hour at room temperature to minimize background while preserving epitope accessibility.

• Primary antibody concentration: Initiate titration at 0.1-0.2 μg/mL and adjust based on signal-to-noise ratio. Data indicates that this antibody remains effective even at higher dilutions (1:5000) .

• Secondary antibody selection: Use HRP-conjugated goat anti-mouse IgG at approximately 0.4 μg/mL (1:2,500 dilution) for optimal signal generation without background interference .

• Detection system: Enhanced chemiluminescence (ECL) systems provide appropriate sensitivity for most applications, though more sensitive substrates may be required for low-abundance tagged proteins.

Western blot analysis performed according to these parameters has successfully detected HA-tagged proteins of various molecular weights (17-77 kDa) in transfected cell lysates, demonstrating the versatility of this antibody across different protein targets .

What methodological approaches enable effective immunoprecipitation of protein complexes using HA-tag antibodies?

Effective immunoprecipitation (IP) of protein complexes using HA-tag antibodies requires careful methodological consideration to maintain complex integrity while achieving high specificity. Research indicates that 1-4 μg of antibody per 100 μg of cell lysate typically provides optimal results . The following methodology has been validated for successful complex isolation:

• Lysate preparation: Utilize non-denaturing lysis buffers containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40 or Triton X-100, supplemented with protease and phosphatase inhibitors. Gentle cell disruption techniques preserve native protein interactions.

• Pre-clearing: Incubate lysates with protein A/G beads for 1 hour at 4°C with gentle rotation to reduce non-specific binding.

• Antibody incubation: Add optimized amounts of HA-tag antibody to pre-cleared lysates and incubate overnight at 4°C with gentle rotation to allow complete antigen-antibody complex formation.

• Bead capture: Add fresh protein A/G beads and incubate for 1-3 hours at 4°C, followed by multiple gentle washes with decreasing salt concentrations to remove non-specifically bound proteins while preserving specific interactions.

• Elution strategies: For analytical applications, standard SDS sample buffer elution at 95°C for 5 minutes is effective. For functional studies, competitive elution with HA peptide (100-200 μg/mL) preserves protein activity by avoiding denaturation .

This approach has successfully isolated complexes containing HA-tagged proteins such as LRRN5 and MICA from transfected human cell lines, demonstrating its efficacy for studying protein-protein interactions under near-physiological conditions .

What controls should be implemented when using HA-tag Peptide PAT5E7AT Antibody for immunofluorescence studies?

Implementing appropriate controls is critical for reliable immunofluorescence studies using the HA-tag Peptide PAT5E7AT Antibody. Comprehensive experimental design should include the following control measures:

• Untransfected control cells: Essential for establishing baseline autofluorescence and determining potential non-specific binding of primary or secondary antibodies to endogenous cellular components .

• Primary antibody omission: Samples processed with secondary antibody only help identify background fluorescence originating from non-specific secondary antibody binding .

• Isotype control: Parallel staining with irrelevant antibodies of the same isotype (Mouse IgG1) at equivalent concentrations helps distinguish between specific binding and Fc receptor-mediated interactions.

• Peptide competition: Pre-incubation of the antibody with excess HA peptide should abolish specific staining, confirming epitope specificity.

• Subcellular localization controls: Include known markers for specific cellular compartments (nuclear, cytoplasmic, membrane) to validate expected localization patterns of the HA-tagged protein.

Empirical data from immunofluorescence experiments demonstrates that effective concentrations of HA-tag antibodies typically range from 2-25 μg/mL depending on the specific application and detection system . Secondary antibodies conjugated to Alexa Fluor dyes (e.g., Alexa Fluor Plus 488) at dilutions of approximately 1:2,000 provide excellent signal-to-noise ratios when paired with appropriate nuclear counterstains like DAPI .

What are the most common causes of false negative results when using HA-tag antibodies, and how can researchers address them?

False negative results with HA-tag antibodies can stem from multiple technical and biological factors. Analysis of research data reveals several common causes and their respective solutions:

• Epitope masking: The three-dimensional protein structure may conceal the HA tag, preventing antibody access. This can be addressed by:

  • Testing alternative fixation protocols that better preserve epitope accessibility

  • Employing different detergents (0.1-0.5% Triton X-100, 0.1% SDS, or 0.5% NP-40) to enhance permeabilization

  • Implementing heat-mediated or enzymatic antigen retrieval methods

• Low expression levels: HA-tagged proteins expressed at low levels may fall below detection thresholds. Solutions include:

  • Increasing protein loading (60-90 μg for Western blots)

  • Using more sensitive detection systems such as enhanced chemiluminescence substrates

  • Implementing signal amplification strategies through tertiary detection systems

• Degradation of tagged proteins: Proteolytic cleavage may remove the HA tag from the protein of interest. Researchers should:

  • Add protease inhibitor cocktails to all extraction buffers

  • Maintain samples at 4°C throughout processing

  • Consider alternative tag positions if C-terminal degradation is suspected

• Competition from endogenous proteins: In some systems, endogenous proteins may competitively bind to the target protein. Researchers can:

  • Use cell lines with lower expression of potential competing proteins

  • Increase stringency of washing steps to remove weak interactions

  • Implement crosslinking approaches to stabilize specific interactions

Comparative analysis indicates that optimizing antibody concentration (typically 0.1-0.2 μg/mL for Western blotting) and incubation conditions (overnight at 4°C) significantly improves detection sensitivity across multiple experimental systems .

How can researchers distinguish between specific and non-specific binding when using the HA-tag Peptide PAT5E7AT Antibody?

Distinguishing between specific and non-specific binding is a critical aspect of experimental validation when using HA-tag antibodies. Evidence-based approaches include:

• Parallel analysis of positive and negative controls: Comparing signals between samples expressing the HA-tagged protein and untransfected controls provides the foundation for specificity assessment. Specific binding should yield clear signals exclusively in positive samples at the predicted molecular weight .

• Peptide competition assays: Pre-incubating the antibody with synthetic HA peptide (YPYDVPDYA) at various concentrations (10-100 μg/mL) should progressively reduce specific signal while leaving non-specific binding unaffected. Quantitative analysis of signal reduction as a function of competing peptide concentration enables precise discrimination between specific and non-specific interactions .

• Signal patterns analysis: Specific binding typically produces consistent patterns across replicates with signal intensities proportional to expression levels. Non-specific binding often presents as inconsistent background or bands that don't correlate with expression levels of the tagged protein.

• Cross-validation with alternative detection methods: Confirming results using secondary detection methods such as mass spectrometry or alternative anti-HA antibody clones provides robust validation. Research indicates that comparing results from at least two independent detection methods significantly enhances confidence in specificity .

• Titration analysis: Serial dilution of the primary antibody typically affects specific signals in a predictable manner, while non-specific binding often shows irregular response patterns to antibody dilution .

Implementation of these methodological approaches has demonstrated that the PAT5E7AT antibody exhibits high specificity when used at recommended concentrations, with minimal cross-reactivity to endogenous cellular proteins in properly optimized systems .

What strategies can improve detection of conformationally hindered HA tags in complex protein structures?

Detecting HA tags within complex protein structures presents unique challenges when epitopes become conformationally hindered. Research-based strategies to overcome these obstacles include:

• Denaturation optimization: Modifying traditional SDS-PAGE conditions by adjusting reducing agent concentration and heating duration can expose hidden epitopes. Empirical testing of varying β-mercaptoethanol concentrations (1-10%) and denaturation temperatures (37-95°C) enables researchers to identify optimal conditions for specific proteins .

• Alternative fixation protocols: For immunocytochemistry, comparing cross-linking fixatives (paraformaldehyde, glutaraldehyde) with precipitating fixatives (methanol, acetone) can identify methods that better preserve epitope accessibility while maintaining adequate structural integrity. Evidence suggests that 4% paraformaldehyde fixation for 10 minutes followed by 0.1% Triton X-100 permeabilization for 15 minutes works effectively for many cellular systems .

• Linker sequence implementation: Introducing flexible glycine-serine linkers (GGGGS)n between the protein of interest and the HA tag increases tag exposure. Experimental data indicates that linkers of 5-15 amino acids significantly improve detection efficiency without substantially altering protein function .

• Tag positioning optimization: Systematic comparison of N-terminal, C-terminal, and internal tagging positions can identify arrangements that maximize epitope accessibility. Research demonstrates that both N-terminal and C-terminal HA tags can be effectively detected by PAT5E7AT antibody, though accessibility varies by protein .

• Chemical modification approach: Mild reduction and alkylation of samples before immunodetection can expose hidden epitopes in cysteine-rich proteins by disrupting disulfide bonds that may conceal the HA tag.

These approaches have successfully improved detection of challenging proteins including transmembrane receptors, nuclear proteins, and large multi-domain proteins where standard protocols yielded suboptimal results .

How does the HA tag compare with other common epitope tags in terms of detection sensitivity and specificity?

Quantitative comparison of epitope tags reveals important performance differences that impact experimental design decisions. Comprehensive analysis of detection sensitivity and specificity across common epitope tags provides valuable insights:

Contrary to common assumptions, rationally designed epitopes (like DYKDDDDK) did not consistently outperform natural epitopes (like HA) in terms of detection sensitivity or specificity. Similarly, newer antibodies did not necessarily generate stronger or more specific signals than established ones . For researchers initiating new projects, both HA and DYKDDDDK tags represent sound choices based on comprehensive performance metrics across multiple experimental systems.

What are the advantages and limitations of HA tags compared to other epitope tags in different experimental contexts?

The decision to use HA tags versus alternative epitope tags should be based on careful consideration of their respective advantages and limitations across different experimental contexts:

Advantages of HA tags:

• Small size (9 amino acids) minimizes interference with protein folding and function, particularly advantageous for smaller proteins or those with critical functional domains .

• Well-characterized epitope with highly specific antibodies available, including PAT5E7AT, providing reliable detection across diverse experimental systems .

• Effective in both N-terminal and C-terminal positions, offering flexibility in construct design based on protein structure considerations .

• Established history in the literature facilitates comparison with previously published results using the same tag system .

• Free from intellectual property restrictions (unlike some newer designed tags), allowing unrestricted production and use in academic and commercial settings .

Limitations:

• Potential cross-reactivity with influenza proteins in certain research contexts involving viral systems.

• May exhibit slightly lower affinity than some newer designed epitopes under specific experimental conditions.

• Less effective for purification applications compared to affinity tags like His6, though still functional for immunoprecipitation .

• Possible recognition by endogenous antibodies in certain immunological studies involving human samples.

When should researchers consider dual-tagging strategies incorporating HA tags with other epitope or affinity tags?

Dual-tagging strategies that combine HA tags with complementary epitope or affinity tags offer significant methodological advantages in complex research applications. Analysis of experimental data supports implementing dual-tagging approaches in the following contexts:

• Sequential purification protocols: Combining HA with orthogonal affinity tags (His6, GST, MBP) enables tandem affinity purification, dramatically reducing contaminants and non-specific interactions. This approach has demonstrated >95% purity in single-step isolations from complex cellular lysates .

• Validation of protein-protein interactions: Using HA tags alongside complementary epitope tags (FLAG, Myc) for reciprocal co-immunoprecipitation provides stringent validation of interaction specificity. This approach helps distinguish true interactions from technical artifacts or antibody cross-reactivity .

• Differential localization studies: Combining N-terminal and C-terminal tags (e.g., N-terminal HA with C-terminal GFP) enables simultaneous tracking of protein processing events, particularly valuable for transmembrane proteins or those undergoing proteolytic processing.

• Quantitative binding studies: Dual tagging facilitates normalization strategies in quantitative binding assays, where one tag (HA) serves for detection/quantification while another (His6) functions for immobilization or purification .

• Expression level optimization: Combining HA with destabilizing tags (PEST sequences) or inducible degrons provides precise control over protein expression levels, particularly valuable for studying dose-dependent effects of regulatory proteins.

Experimental evidence indicates that careful positioning of dual tags is critical to prevent interference between tags or with protein function. Strategic placement of flexible linker sequences (5-15 amino acids) between the protein and each tag significantly improves detection efficiency and functional preservation . For smaller proteins (<30 kDa), researchers should be particularly attentive to potential structural impacts of multiple tags and may consider alternative strategies such as split tag approaches.

What are the key considerations for selecting appropriate HA-tag antibodies for specific research applications?

Selecting the appropriate HA-tag antibody requires systematic evaluation of multiple parameters aligned with specific research objectives. Based on comprehensive analysis of experimental evidence, researchers should consider:

• Application compatibility: Different antibody clones demonstrate variable performance across applications. The PAT5E7AT antibody has been validated for Western blot, immunoprecipitation, and immunocytochemistry applications . Researchers should verify performance data for their specific application.

• Sensitivity requirements: For low abundance proteins, antibodies demonstrating high sensitivity at lower concentrations (e.g., AF291) may be preferable . Comparative testing may be necessary for extremely challenging detection scenarios.

• Format considerations: Available formats (unconjugated, directly conjugated, solution vs. lyophilized) influence workflow design. The PAT5E7AT antibody is typically supplied as a sterile filtered colorless solution , which offers convenience for most standard applications.

• Cross-reactivity profile: Evaluate potential cross-reactivity with endogenous proteins, particularly in specialized cell types or when studying influenza-related proteins where natural epitope similarity may exist.

• Reproducibility considerations: Well-established antibodies with consistent manufacturing processes typically offer superior lot-to-lot reproducibility for longitudinal studies.

• Species compatibility: While most anti-HA antibodies recognize the tag independently of the host species expressing the construct, secondary antibody selection must align with the primary antibody's host species (mouse for PAT5E7AT) .

Through careful evaluation of these parameters against specific research requirements, investigators can select appropriate antibodies that maximize experimental success while minimizing technical challenges and resource expenditure.

How has the development of HA-tag antibody technology impacted modern molecular biology research?

The development and refinement of HA-tag antibody technology has profoundly influenced modern molecular biology research through several transformative impacts:

• Democratization of protein analysis: HA-tag antibodies have enabled protein detection and characterization without requiring protein-specific antibodies, particularly valuable for newly discovered or poorly immunogenic proteins . This has significantly accelerated research timelines and reduced resource requirements.

• Standardization of detection methods: The widespread adoption of HA-tag systems has facilitated methodological standardization across research groups, enhancing reproducibility and enabling more direct comparison of results across studies and laboratories .

• Expansion of multiplexed analysis: HA tags in combination with other epitope tags have enabled simultaneous tracking of multiple proteins within the same cellular system, driving advances in systems biology approaches to understanding complex protein interaction networks .

• Enhancement of quantitative proteomics: HA-tag technologies have contributed to the development of more precise quantitative methods for protein analysis, including competition assays and normalization strategies that improve accuracy by 4- to 10-fold compared to traditional approaches .

• Facilitation of structural biology: By providing reliable purification methods with minimal impact on protein structure, HA-tag technologies have contributed to significant advances in protein structure determination, particularly for challenging targets.