HERC5 Antibody, HRP conjugated

What is HERC5 and why is it significant in immunological research?

HERC5 (Hect Domain and RLD 5) is a critical protein involved in the regulation of the interferon pathway, a key component of the innate immune response against viral infections. Its significance stems from its role in activating interferon-stimulated genes (ISGs), which are essential for antiviral defense mechanisms and immune surveillance. HERC5 functions as an E3 ISG15-protein ligase, contributing to the post-translational modification process called ISGylation. Research into HERC5 provides valuable insights into host-virus interactions, immune system regulation, and potential therapeutic targets for viral diseases . The protein has been shown to restrict various viruses by modifying viral proteins through ISG15 conjugation, making it a crucial component in understanding cellular defense mechanisms against pathogens.

What are the advantages of using HRP-conjugated HERC5 antibodies over unconjugated versions?

HRP (horseradish peroxidase)-conjugated HERC5 antibodies offer several significant advantages in research applications. The direct conjugation eliminates the need for secondary antibodies, which simplifies experimental workflows, reduces background noise, and decreases the potential for cross-reactivity. This streamlined approach is particularly valuable in multi-color immunoassays where multiple primary antibodies are used simultaneously. HRP-conjugated antibodies like ABIN7378048 provide enhanced sensitivity in ELISA applications through enzymatic signal amplification, which can detect smaller quantities of target protein . Additionally, these conjugated antibodies reduce the total incubation time required for immunoassays, allowing for faster experimental turnaround while maintaining specificity for their target epitopes, such as amino acids 153-284 in HERC5 .

What is the molecular weight and structure of HERC5, and why is this relevant for antibody selection?

HERC5 is a large protein with a calculated molecular weight of approximately 117 kDa, comprising 1024 amino acids . The protein contains multiple functional domains, including the HECT domain (responsible for E3 ligase activity) and RCC1-like domains (RLDs) that mediate protein-protein interactions. When selecting HERC5 antibodies, researchers must consider the specific epitope recognized by the antibody. Different antibodies target distinct regions of HERC5, such as the N-terminus, middle region (amino acids 153-284, 287-336), or C-terminal regions (amino acids 900-949, 915-1024) . This epitope specificity is crucial as protein folding, post-translational modifications, or protein-protein interactions may mask certain epitopes in experimental conditions. For example, when studying HERC5 in complex with ISG15 or target proteins, antibodies targeting the HECT domain might show reduced binding if this domain is occluded in the complex.

How should I determine the optimal dilution of HRP-conjugated HERC5 antibodies for Western blotting?

Determining the optimal dilution for HRP-conjugated HERC5 antibodies requires a systematic titration approach. Begin with a broad dilution range based on manufacturer recommendations (typically 1:500-1:3000 for Western blotting) . Perform a dilution series experiment using the same amount of positive control lysate (such as from HEK-293 cells known to express HERC5) across multiple membrane strips. Process all strips identically except for primary antibody concentration. Evaluate signal-to-noise ratio, background levels, and specific band intensity at the expected molecular weight (117 kDa for HERC5) . The optimal dilution provides clear specific bands with minimal background. Important considerations include: (1) extending incubation time may allow for more dilute antibody solutions; (2) optimization should be repeated when changing detection systems; and (3) sample-dependent factors might necessitate reoptimization when studying different tissues or experimental conditions . Document your optimization process methodically to ensure reproducibility across experiments.

What is the recommended protocol for using HRP-conjugated HERC5 antibodies in ELISA assays?

For optimal ELISA performance using HRP-conjugated HERC5 antibodies like ABIN7378048, follow this methodological approach: Begin by coating high-binding ELISA plates with capture antibody or antigen (depending on sandwich or direct ELISA format) overnight at 4°C. After blocking with 3-5% BSA or non-fat milk in PBST for 1-2 hours at room temperature, add samples and standards at appropriate dilutions. For detection, apply the HRP-conjugated HERC5 antibody at the optimal dilution as determined through titration experiments . The exact working dilution should be empirically determined for each new experimental system rather than relying solely on manufacturer recommendations. Incubate for 1-2 hours at room temperature followed by 4-6 thorough washes with PBST. Develop with TMB substrate and stop the reaction with 2N H₂SO₄ when appropriate color development occurs. When measuring optical density, 450nm is the primary wavelength, with 570nm serving as a reference wavelength for background subtraction. Critical considerations include maintaining consistent incubation times across plates and running all standards and samples in at least duplicate.

How do I troubleshoot weak or non-specific signals when using HRP-conjugated HERC5 antibodies in Western blots?

When encountering weak or non-specific signals with HRP-conjugated HERC5 antibodies in Western blots, implement a systematic troubleshooting approach. For weak signals: (1) Verify HERC5 expression in your samples using published literature on expression patterns; (2) Decrease antibody dilution while monitoring background; (3) Extend primary antibody incubation time to overnight at 4°C; (4) Increase protein loading but ensure it doesn't exceed 50 μg per lane to prevent smearing; (5) Enhance detection sensitivity using extended exposure times or more sensitive substrates. For non-specific bands: (1) Optimize blocking conditions by testing alternative blocking agents (5% BSA often performs better than milk for phospho-specific antibodies); (2) Increase washing stringency by adding 0.1% SDS or increasing salt concentration in wash buffers; (3) Preabsorb antibody with non-specific proteins; (4) Validate bands using positive controls with known HERC5 expression and molecular weight (117 kDa) ; (5) Consider using validated HERC5 knockout samples as negative controls to identify specific bands . Documentation of optimization steps is crucial for reproducibility and troubleshooting across experiments.

Why might there be discrepancies in results between different applications (WB, IHC, ELISA) using the same HERC5 antibody?

Discrepancies in results across different applications using the same HERC5 antibody stem from fundamental methodological differences in how proteins are presented to antibodies. In Western blotting, proteins are denatured and linearized, exposing epitopes that might be hidden in native conformations. In contrast, immunohistochemistry (IHC) and ELISA often maintain proteins in more native states, where three-dimensional folding can mask certain epitopes. The HERC5 antibody's performance across these methods depends on:

• Epitope accessibility: The amino acid sequence targeted by the antibody (e.g., aa 153-284 or 360-700) may be differently exposed in each method

• Fixation effects: Formalin fixation in IHC can modify epitopes through protein cross-linking

• Buffer compatibility: Some antibodies perform optimally in specific buffer conditions that vary between applications

• Concentration requirements: Different applications typically require distinct antibody concentrations

This explains why manufacturers often validate HERC5 antibodies for specific applications rather than assuming cross-application functionality. For example, ABIN7464280 is specifically validated for Western blotting , while others might be optimized for multiple applications including ELISA, IHC, and IF . When encountering discrepancies, researchers should consider whether the epitope's presentation fundamentally differs between methods.

How can I determine if my experimental results with HERC5 antibodies are affected by cross-reactivity with other HERC family proteins?

Cross-reactivity between HERC family proteins (HERC1-6) presents a significant challenge for HERC5 antibody specificity due to sequence homology, particularly in conserved domains. To assess and mitigate cross-reactivity issues, implement these methodological approaches:

• Bioinformatic analysis: Compare the antibody's target sequence (e.g., amino acids 360-700 of HERC5) against all HERC family proteins using sequence alignment tools to identify potential cross-reactive regions.

• Validation using knockout/knockdown models: Utilize HERC5 knockout cell lines or siRNA-mediated knockdown to confirm signal specificity. A true HERC5-specific antibody should show significantly reduced signal in these models.

• Cross-absorption controls: Pre-incubate your HERC5 antibody with recombinant HERC family proteins to determine if this reduces signal in your experimental system.

• Immunoprecipitation-mass spectrometry: Perform IP with your HERC5 antibody followed by mass spectrometry to identify all captured proteins, revealing potential cross-reactivity.

• Parallel testing with multiple HERC5 antibodies: Compare results using antibodies targeting different HERC5 epitopes (N-terminal, middle region, C-terminal) . Consistent results across antibodies targeting distinct regions increase confidence in specificity.

This methodical approach allows for rigorous assessment of cross-reactivity and appropriate interpretation of experimental results.

How can HRP-conjugated HERC5 antibodies be utilized in multiplexed immunoassays for studying interferon response pathways?

HRP-conjugated HERC5 antibodies offer sophisticated capabilities for multiplexed analysis of interferon response pathways. A methodological approach for such studies would involve strategic antibody pairing and signal differentiation. Begin by selecting HRP-conjugated HERC5 antibodies (like ABIN7378048) alongside complementary antibodies against other ISG pathway components (ISG15, USP18, etc.) conjugated to different enzymes or fluorophores. This allows simultaneous detection of multiple targets in a single sample.

For chromogenic multiplexing, implement sequential immunostaining with spectrally distinct substrates for each enzyme (DAB for HRP, Fast Red for AP). Between staining rounds, perform antibody stripping or inactivation using either heat treatment (95°C for 5 minutes in citrate buffer) or chemical methods (glycine-HCl buffer, pH 2.5).

For fluorescent multiplexing, utilize tyramide signal amplification (TSA) with the HRP-conjugated HERC5 antibody, which provides 10-200 fold signal enhancement through covalent deposition of fluorescent tyramide. This approach allows detection of low-abundance HERC5 protein while preserving the ability to multiplex with other markers.

Critical considerations include: (1) optimizing antibody concentration to prevent signal bleed-through; (2) including proper controls for non-specific binding; and (3) employing image analysis software capable of spectral unmixing to resolve overlapping signals in multiplexed assays.

What are the considerations for using HERC5 antibodies in studying post-translational modifications and protein-protein interactions?

Investigating post-translational modifications (PTMs) and protein-protein interactions (PPIs) of HERC5 requires careful antibody selection and methodological considerations. When studying PTMs, researchers must determine whether the antibody's epitope (e.g., amino acids 153-284 or 360-700) contains or is adjacent to known modification sites. If the PTM occurs within the epitope, it may block antibody binding, resulting in false negatives. For HERC5's E3 ligase activity studies, use paired antibodies targeting different epitopes—one recognizing the HECT domain and another distant from it—to confirm results.

For protein-protein interaction studies, consider these methodological approaches:

• Co-immunoprecipitation validation: When using HERC5 antibodies for Co-IP , verify that the antibody doesn't compete with interaction partners for binding to HERC5. Control experiments should include:

  • IP with non-specific IgG

  • Reverse IP using antibodies against the suspected binding partner

  • Competition assays with recombinant proteins

• Proximity ligation assays (PLA): This technique can visualize HERC5 interactions in situ with high specificity. Combine unconjugated HERC5 antibodies with antibodies against potential interaction partners, followed by species-specific secondary antibodies conjugated to complementary oligonucleotides.

• FRET/BRET approaches: These techniques require careful antibody fragment conjugation to avoid steric hindrance that might disrupt natural protein interactions.

The experimental design should account for the large size of HERC5 (117 kDa) and its multi-domain structure, which may result in complex interaction profiles across different cellular compartments.

How can HERC5 antibodies be employed in high-content screening approaches to identify novel antiviral compounds?

High-content screening (HCS) utilizing HERC5 antibodies presents a sophisticated approach to identifying novel antiviral compounds that modulate ISGylation pathways. A methodologically sound HCS campaign would incorporate the following elements:

• Assay development and validation:

  • Establish stable cell lines expressing endogenous or tagged HERC5

  • Optimize HRP-conjugated or fluorescently labeled HERC5 antibodies for cellular imaging

  • Develop quantitative parameters including HERC5 expression levels, subcellular localization, and colocalization with viral proteins

• Screening methodology:

  • Implement automated imaging in 384-well format

  • Pre-treat cells with compound libraries followed by viral infection or interferon stimulation

  • Fix, permeabilize, and immunostain using validated HERC5 antibodies targeting appropriate epitopes (e.g., aa 153-284)

  • Include positive controls (known HERC5 modulators) and negative controls (vehicle)

• Multiparametric analysis:

  • Quantify HERC5 expression levels

  • Assess changes in subcellular localization

  • Measure colocalization with ISGylated targets

  • Correlate with viral replication markers

• Hit validation pipeline:

  • Confirm hits using orthogonal assays (Western blot, qPCR)

  • Establish dose-response relationships

  • Determine mechanism of action through Co-IP and mass spectrometry

This approach allows for identification of compounds that specifically modulate HERC5 activity rather than general interferon pathway activators, potentially leading to more targeted antiviral therapies with fewer side effects.

What are the key differences between various commercially available HERC5 antibodies in terms of epitope recognition and validation?

Commercial HERC5 antibodies exhibit significant differences in epitope targeting, validation methods, and performance across applications. This comparative analysis reveals distinct characteristics that impact experimental design and result interpretation:

Key comparative insights:

• Epitope variability: Different antibodies target distinct regions of HERC5, from N-terminal to C-terminal domains, affecting their utility in different experimental contexts

• Validation depth: More extensively validated antibodies like 22692-1-AP with published studies provide higher confidence

• Application specificity: Some antibodies are validated for multiple applications (e.g., 22692-1-AP) , while others are optimized for specific techniques like ELISA (ABIN7378048)

• Conjugation options: HRP-conjugated options (ABIN7378048) offer direct detection capabilities, while unconjugated versions provide flexibility

These differences underscore the importance of selecting HERC5 antibodies based on specific experimental requirements rather than assuming equivalent performance across all research contexts.

How do variations in sample preparation affect the detection sensitivity of HERC5 using HRP-conjugated antibodies?

Sample preparation methodology significantly impacts HERC5 detection sensitivity when using HRP-conjugated antibodies. This relationship stems from how preparation techniques affect protein structure, epitope accessibility, and background signal:

• Lysis buffer composition:

  • RIPA buffer provides efficient extraction but may partially denature HERC5, affecting epitope recognition

  • NP-40 or Triton X-100 based buffers better preserve protein-protein interactions but may yield lower extraction efficiency

  • Optimization data indicates adding 1-10 mM N-ethylmaleimide (NEM) to lysis buffers preserves ISGylation and HERC5-substrate interactions

• Protein denaturation conditions:

  • Complete denaturation (boiling with SDS/DTT) maximizes linear epitope exposure (beneficial for antibodies like ABIN7378048 targeting aa 153-284)

  • Native conditions preserve conformational epitopes and protein complexes

  • Empirical testing shows 70°C for 10 minutes often provides optimal balance for HERC5 detection

• Enrichment strategies:

  • Immunoprecipitation before detection can significantly enhance sensitivity for low-abundance HERC5 (recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg lysate)

  • Subcellular fractionation can concentrate HERC5 from relevant compartments

• Cross-linking considerations:

  • Formaldehyde fixation (common in IHC/ICC) may mask the epitope recognized by certain HERC5 antibodies

  • Epitope retrieval methods should be optimized specifically for HERC5 detection

Researchers should conduct parallel processing of identical samples using different preparation methods to determine optimal conditions for their specific HRP-conjugated HERC5 antibody and experimental system.

What advanced imaging techniques can be combined with HERC5 antibodies to study its dynamic localization during viral infection?

Advanced imaging techniques coupled with HERC5 antibodies enable sophisticated analysis of its dynamic localization during viral infection. Methodological approaches include:

• Live-cell super-resolution microscopy:

  • Implement STED (Stimulated Emission Depletion) or PALM (Photoactivated Localization Microscopy) with HERC5 antibody fragments conjugated to appropriate fluorophores

  • This approach achieves 20-50 nm resolution, revealing HERC5 redistribution during viral infection that conventional microscopy would miss

  • Time-lapse imaging captures dynamic HERC5 trafficking between cellular compartments

• Correlative Light and Electron Microscopy (CLEM):

  • Initially locate HERC5 via fluorescently labeled antibodies in light microscopy

  • Subsequently examine the same sample with electron microscopy

  • This technique precisely maps HERC5 localization to ultrastructural features altered during viral infection

• Fluorescence Recovery After Photobleaching (FRAP):

  • When combined with fluorescently labeled HERC5 antibodies, FRAP measures HERC5 mobility

  • Differential mobility parameters before and after viral infection indicate functional changes

• Proximity Ligation Assay (PLA):

  • Using paired antibodies against HERC5 and viral proteins

  • Generates fluorescent signals only when proteins are within 40 nm

  • Provides spatial and temporal mapping of HERC5-viral protein interactions

• Lattice Light-Sheet Microscopy:

  • Offers minimal phototoxicity for extended imaging

  • Captures rapid HERC5 redistribution events at subcellular resolution over hours of viral infection