HERC5 Antibody, Biotin conjugated

Basic Research Questions

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

HERC5 (HECT domain and RLD 5) is the main ISG15 E3 ligase in human cells that conjugates ISG15 to cellular proteins during the interferon response. It functions in concert with E1 activating protein Ube1L and E2 conjugating protein UbcH8 to facilitate ISGylation . HERC5 possesses several key structural characteristics:

• Contains RCC1-like repeats at the N-terminus (residues 209-258)

• Features a HECT domain at the C-terminus (residues 676-1024)

• Includes a conserved cysteine residue (C994) essential for E3 ligase activity

HERC5 is strongly induced by type I interferons, with mRNA levels increasing approximately 30-fold in both HeLa and A549 cells after 12 hours of IFN-β treatment . This interferon-induced protein has gained significant attention for its role in viral restriction, particularly against HIV-1 replication through multiple mechanisms including inhibition of Gag particle assembly and targeting Rev/RRE-dependent RNA nuclear export .

• What are the optimal applications for biotin-conjugated HERC5 antibodies?

Based on available research data, biotin-conjugated HERC5 antibodies are particularly valuable for:

• ELISA applications: The biotin-streptavidin interaction significantly enhances detection sensitivity through signal amplification

• Multiplex immunoassays: Biotin conjugation allows for simultaneous detection of multiple targets using different reporter systems

• Protein interaction studies: Especially useful for investigating HERC5's interactions with polyribosomes, HIV-1 Gag, and the ISGylation machinery

• Pull-down assays: The strong biotin-streptavidin bond (Kd ≈ 10^-15 M) facilitates efficient isolation of HERC5-containing complexes

While unconjugated HERC5 antibodies are commonly used for Western blot, immunoprecipitation, and immunohistochemistry applications , the biotin-conjugated versions offer enhanced sensitivity and versatility for specific experimental setups where signal amplification is crucial.

• How should researchers optimize interferon treatment for HERC5 detection?

Optimizing interferon treatment is critical for successful HERC5 detection due to its interferon-inducible nature:

The kinetics demonstrate that HERC5 mRNA expression precedes detectable protein activity . For optimal experimental design:

• Use 250-1000 U/ml of type I interferon (particularly IFN-β)

• Include time course analysis if studying dynamic ISGylation processes

• For detection of endogenous HERC5 in untransfected cells, 24-48 hour interferon treatment is strongly recommended

• Consider cell type variations in interferon responsiveness when planning experiments

• What controls are essential when using biotin-conjugated HERC5 antibodies?

Essential controls for experiments using biotin-conjugated HERC5 antibodies include:

• Genetic controls: HERC5 knockout or knockdown cells (via CRISPR or shRNA) to validate signal specificity

• Structural controls: HERC5-C994A mutant (inactive E3 ligase) to distinguish enzyme-dependent from enzyme-independent effects

• Technical controls:

  • Streptavidin-only binding assessment

  • Non-specific biotinylated IgG of matching isotype

  • Blocking experiments with excess unlabeled antibody

• Biological controls:

  • Interferon-treated versus untreated samples

  • Comparison with other members of the HERC family (particularly HERC6, the closest homolog)

The shRNA-HERC5-1-4 construct targeting the 1,606-1,639 bp region of HERC5 has been demonstrated to effectively reduce HERC5 expression without affecting HERC6 levels, making it a useful control tool .

Advanced Research Questions

• How can biotin-conjugated HERC5 antibodies be optimized for detecting ISGylation events in proteomics workflows?

For proteomics-based ISGylation studies using biotin-conjugated HERC5 antibodies:

Sample preparation optimization:

• Pre-treat cells with IFN-β for 48 hours to maximize ISGylation machinery expression

• Process total cell lysates with purified USP2-cc (catalytic core), which reduces ubiquitin conjugates by approximately 85% without affecting ISG15 conjugates

• Use non-denaturing lysis conditions followed by appropriate denaturation before immunoprecipitation

Enrichment strategy:

• Perform sequential immunoprecipitation: first with anti-K-ε-GG antibodies to capture diglycine-modified peptides, then with biotin-conjugated HERC5 antibodies

• Utilize streptavidin-coated magnetic beads for efficient capture of biotin-antibody-protein complexes

Analysis approach:

• Apply hierarchical clustering to identified modification sites to distinguish:

  • Cluster 1: Sites present across both wild-type and HERC5-KO samples (non-specific or HERC5-independent modifications)

  • Cluster 2: Sites specific to wild-type samples (approximately 2,500 sites) representing HERC5-dependent ISGylation

  • Cluster 3: Low-reproducibility sites (discard from analysis)

• Define HERC5-dependent ISG15 modification sites as those identified in at least two of three biological replicates in interferon-treated cells but absent in HERC5-KO samples

This methodology successfully identified HERC5-dependent ISGylation events in previous research and can be adapted for various experimental designs .

• What methodological approaches can improve visualization of HERC5's association with polyribosomes?

To effectively visualize HERC5's association with polyribosomes:

Confocal immunofluorescence approach:

• Treat cells with interferon-β to induce HERC5 expression

• Use biotin-conjugated HERC5 antibodies with streptavidin-fluorophore detection

• Co-stain with ribosomal markers (e.g., antibodies against RPL7 or other ribosomal proteins)

• Apply appropriate fixation methods that preserve polyribosome structures

• Analyze co-localization quantitatively using Pearson's coefficient measurements

Previous studies demonstrated that 59.9% ± 0.12% SD of HERC5 co-localized with polyribosomes with a mean Pearson's coefficient of 0.855 , providing a benchmark for successful visualization.

Biochemical confirmation:

• Perform polysome fractionation using sucrose gradient ultracentrifugation

• Analyze fractions by Western blotting using biotin-conjugated HERC5 antibodies

• Include controls with:

  • EDTA treatment (disrupts polysomes)

  • RNase treatment (degrades RNA component)

  • HERC5 mutants lacking the N-terminal RCC1-like domain (required for ribosome association)

For super-resolution approaches, consider using techniques such as Stimulated Emission Depletion (STED) microscopy to achieve higher resolution of HERC5-polyribosome interactions at the nanoscale level.

• How should researchers design experiments to study HERC5's interaction with viral proteins using biotin-conjugated antibodies?

When investigating HERC5's interaction with viral proteins such as HIV-1 Gag:

Co-immunoprecipitation protocol:

• Use non-denaturing lysis conditions to preserve protein-protein interactions

• Perform reciprocal co-immunoprecipitation:

  • Immunoprecipitate with anti-p24CA (Gag) and detect HERC5

  • Immunoprecipitate with biotin-conjugated HERC5 and detect Gag

• Include HERC5-C994A mutant control to distinguish E3 ligase-dependent from independent interactions

Microscopy-based approach:

• Co-express HERC5 with viral proteins (e.g., HIV-1 Gag)

• Analyze optical slices through the center of cells for accurate co-localization assessment

• Quantify co-localization using statistical measures:

  • Previous studies found 65.2% ± 0.21% SD of Gag co-localized with HERC5 (mean Pearson's coefficient = 0.324)

  • For Gag-only expression, 53.1% ± 0.16% SD co-localization was observed (mean Pearson's coefficient = 0.336)

Functional assessment:

• Use cell-based viral restriction assays to correlate physical interactions with functional outcomes

• Measure viral particle production in the presence of wild-type HERC5 versus HERC5-C994A

• For HIV-1 studies, examine both early assembly stages at the plasma membrane and Rev/RRE-dependent RNA export

These methods have successfully demonstrated HERC5's role in viral restriction and can be adapted for studies with other viral systems.

• What are the optimal conditions for studying HERC5's role in RanGTP-mediated nuclear transport using biotin-conjugated antibodies?

To investigate HERC5's impact on RanGTP-mediated nuclear transport:

Protein interaction studies:

• Lyse cells under non-denaturing conditions to preserve HERC5-Ran interactions

• Perform co-immunoprecipitation using either anti-Ran antibodies or biotin-conjugated HERC5 antibodies

• Include appropriate controls (GTPγS-treated lysates, HERC5 knockout cells)

RanGTP level assessment:

• Utilize the RanBP1-coated agarose bead pull-down assay, which specifically binds RanGTP but not RanGDP

• Process lysates from control cells, vector-transfected cells, and HERC5-expressing cells

• Quantify RanGTP levels using Western blotting with anti-Ran antibodies

Microscopy approach:

• Examine Ran distribution between nucleus and cytoplasm in the presence/absence of HERC5

• Use biotin-conjugated HERC5 antibodies with streptavidin-fluorophore detection

• Combine with fluorescent cargo proteins dependent on Ran-mediated nuclear transport

This experimental approach allows for investigation of HERC5's potential impact on nuclear transport pathways that might contribute to its antiviral functions beyond direct ISGylation of viral proteins.

• How can researchers differentiate between HERC5's E3 ubiquitin ligase and E3 ISG15 ligase activities?

HERC5 has been identified as both an E3 ubiquitin ligase and an E3 ISG15 ligase . To differentiate these activities:

Biochemical differentiation approach:

• Use reconstitution systems with specific E1/E2 enzymes:

  • For ISGylation: UBA7 (E1) and UBE2L6 (E2)

  • For ubiquitination: UBE1 (E1) and appropriate E2s

• Co-express HERC5 with either ISG15 or ubiquitin along with the respective E1/E2 enzymes

• Analyze conjugation products by Western blotting

Mutational analysis:

• The C994A mutation in the HECT domain abolishes both ubiquitin and ISG15 ligase activities

• Domain-specific mutations or truncations may differentially affect ubiquitin versus ISG15 conjugation

Substrate specificity analysis:

• Compare proteins modified by HERC5-dependent ubiquitination versus ISGylation using mass spectrometry

• Previous proteomic studies identified 174 candidate proteins conjugated or interacting with ISG15 upon interferon treatment

• Of 27 targets examined in detail, 24 were confirmed to be conjugated with ISG15

Experimental controls:

• Include Ube1 (ubiquitin E1) knockdown controls to rule out that HERC5's ISG15 ligase activity depends on prior ubiquitination

• Use specific deconjugating enzymes (USP18 for ISG15, various DUBs for ubiquitin) to confirm modification type

Understanding the dual E3 ligase functionality of HERC5 is important for comprehending its full range of cellular activities and potential therapeutic applications.

• What are the critical considerations when designing experiments to study HERC5's evolutionary adaptation to viral pathogens?

To investigate HERC5's evolution as an antiviral factor:

Comparative genomics approach:

• Analyze HERC5 sequences across different species, particularly focusing on:

  • Primate lineages (human, chimpanzee, gorilla, orangutan, etc.)

  • Key functional domains (RCC1-like domain, HECT domain)

  • Sites under positive selection pressure

• Compare with related HERC family members (especially HERC6, which serves as the main ISG15 E3 ligase in mice)

Functional validation:

• Create chimeric HERC5 proteins with domains from different species

• Test the antiviral activity of these chimeras against various viral challenges

• Focus on regions showing signs of positive selection

Experimental controls:

• Include ancestral sequence reconstructions as controls

• Test activity against viruses that co-evolved with the species from which HERC5 sequences were derived

• Include HERC5-C994A catalytic mutants to distinguish E3 ligase-dependent from independent restriction mechanisms

Biotin-conjugated antibody application:

• Use species-specific biotin-conjugated HERC5 antibodies to compare expression patterns and localization across different species

• Ensure antibodies recognize conserved epitopes when comparing across species, or use species-specific antibodies as appropriate

These approaches can help identify how HERC5 has adapted to combat viral pathogens throughout evolutionary history and may reveal novel antiviral mechanisms.