VACWR106 Antibody

What is the VACWR106 Antibody and what is its molecular target?

The VACWR106 Polyclonal Antibody is developed against the VACWR106 protein from Vaccinia virus (VACV), specifically the Western Reserve strain. This antibody targets the mRNA-capping enzyme catalytic subunit, also known as the virus termination factor large subunit (VTF large subunit) or mRNA-capping enzyme 97 kDa subunit . The target protein contains several enzymatic activities essential for viral mRNA processing, including polynucleotide 5'-triphosphatase (EC 3.1.3.33), mRNA guanylyltransferase (EC 2.7.7.50), and mRNA (guanine-N(7)-)-methyltransferase (EC 2.1.1.56) . These enzymatic functions are critical for viral mRNA capping, which protects viral transcripts from degradation and enables efficient translation.

How does the VACWR106 antibody contribute to understanding vaccinia virus biology?

The VACWR106 antibody provides researchers with a valuable tool to investigate the expression, localization, and function of the mRNA capping enzyme in vaccinia virus infection models. This antibody allows for the detection and quantification of the VACWR106 protein, which plays essential roles in viral transcription and mRNA processing. By studying this protein, researchers can better understand fundamental mechanisms of poxvirus gene expression and identify potential targets for antiviral interventions. Immunological studies of vaccinia virus have demonstrated that both cellular and humoral responses target a wide range of antigens and epitopes, with VACWR106 being one of the viral components recognized by the immune system .

What are the general characteristics of antibody responses to vaccinia virus proteins?

Antibody responses to vaccinia virus (VACV) proteins are remarkably broad and diverse. Studies have shown that both cellular and humoral immune responses target a large number of antigens and epitopes across the viral proteome . This broad spectrum of targets is observed in both murine models and human subjects. The antibody response against vaccinia typically includes recognition of both intracellular mature virion (IMV) and extracellular enveloped virion (EEV) antigens . Effective neutralization of the virus requires antibodies targeting both viral particle types, as protection against virus challenge is most effective when a combinatory approach targeting both IMV and EEV is employed .

What validated experimental applications are available for the VACWR106 Antibody?

The VACWR106 Polyclonal Antibody has been validated for specific experimental applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

These applications enable researchers to detect and quantify VACWR106 protein in various experimental contexts. For Western blotting applications, the antibody allows detection of the ~97 kDa protein in viral-infected cell lysates. For ELISA applications, the antibody can be used to quantitatively measure VACWR106 in purified viral preparations or infected cell extracts.

What methodological considerations are important when using antibodies against viral proteins in research?

When using antibodies against viral proteins like VACWR106, several methodological considerations are crucial:

Additionally, researchers should evaluate the thermal stability of antibody preparations, as thermal stability can significantly impact experimental reproducibility. Recent studies on antibody fragments have shown that their melting temperatures (Tms) typically range from approximately 50–70°C, which can affect long-term storage and experimental conditions .

How can researchers optimize immunological detection of VACWR106 in infected cells?

Optimizing the detection of VACWR106 in infected cells requires attention to several experimental parameters:

• Infection timing: Since VACWR106 is involved in viral mRNA capping, optimal detection may require harvesting cells during active viral transcription, typically 4-8 hours post-infection.

• Cell fixation and permeabilization: For immunofluorescence applications, test different fixatives (paraformaldehyde, methanol, acetone) and permeabilization agents (Triton X-100, saponin) to determine optimal conditions for epitope preservation and accessibility.

• Antibody dilution: Perform titration experiments to identify the optimal antibody concentration that maximizes specific signal while minimizing background.

• Signal amplification: Consider using signal amplification techniques such as tyramide signal amplification or polymer-based detection systems to enhance sensitivity.

• Counterstaining: Use appropriate nuclear and cellular markers to provide context for the VACWR106 localization pattern.

For Western blotting applications, researchers should optimize protein extraction methods to ensure efficient solubilization of the viral protein while preserving epitope integrity.

What is known about the specificity of VACWR106 Antibody across different poxvirus species?

The VACWR106 Polyclonal Antibody was developed against the recombinant Vaccinia virus (strain Western Reserve) VACWR106 protein (specifically amino acids 516-844) . While primarily targeting VACV strain WR, researchers should consider potential cross-reactivity with homologous proteins from other poxviruses. Orthopoxviruses such as variola virus (VARV, smallpox), monkeypox virus, and cowpox virus contain highly conserved mRNA capping enzymes that may be recognized by this antibody.

How can researchers assess potential cross-reactivity with host cell proteins?

Assessing cross-reactivity with host cell proteins requires careful experimental design:

• Negative control samples: Include uninfected cell lysates or tissues to identify any non-specific binding to host proteins.

• Competitive binding assays: Pre-incubate the antibody with purified recombinant VACWR106 protein before application to samples to verify that signals are specifically blocked.

• Immunoprecipitation followed by mass spectrometry: This approach can identify all proteins captured by the antibody, revealing potential cross-reacting host proteins.

• Bioinformatic analysis: Compare sequences of the immunogen (amino acids 516-844 of VACWR106) against the host proteome to predict potential cross-reactivity.

• Knockout or knockdown validation: If available, use cells with CRISPR-mediated knockout of cross-reacting host proteins to confirm specificity.

Cross-reactivity assessment is particularly important for polyclonal antibodies like VACWR106 Antibody, as they recognize multiple epitopes and may have greater potential for non-specific binding compared to monoclonal antibodies.

What approaches can determine if VACWR106 Antibody recognizes conformational or linear epitopes?

Understanding whether the VACWR106 Antibody recognizes conformational or linear epitopes is crucial for selecting appropriate experimental conditions. Researchers can employ several approaches:

• Comparison of reducing vs. non-reducing Western blot conditions: If signal is lost under reducing conditions, this suggests recognition of conformational epitopes dependent on disulfide bonds.

• Heat denaturation tests: Compare antibody binding to native vs. heat-denatured antigen to assess dependence on tertiary structure.

• Epitope mapping using peptide arrays: Synthesize overlapping peptides spanning the immunogen sequence (amino acids 516-844) to identify linear epitopes recognized by the antibody.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can identify regions of the antigen that are protected from exchange upon antibody binding, revealing epitopes.

• X-ray crystallography or cryo-EM: While resource-intensive, structural determination of antibody-antigen complexes provides definitive information about epitope characteristics, similar to approaches used to characterize other antibody-antigen interactions .

How can VACWR106 Antibody be utilized to study vaccinia virus mRNA capping mechanisms?

The VACWR106 Antibody provides several sophisticated approaches to investigate viral mRNA capping:

• Immunoprecipitation of capping complexes: The antibody can be used to pull down VACWR106 and associated proteins, enabling identification of viral or host factors that interact with the capping machinery.

• ChIP-seq applications: By performing chromatin immunoprecipitation followed by sequencing, researchers can map the association of VACWR106 with viral DNA during transcription, potentially revealing gene-specific capping dynamics.

• Inhibitor studies: The antibody can be used to monitor changes in VACWR106 localization or complex formation in response to compounds that target viral transcription or capping.

• Microscopy-based colocalization: Combined with antibodies against other viral transcription factors, VACWR106 Antibody enables visualization of viral transcription factory formation and dynamics.

• Depletion experiments: In cell-free transcription systems, the antibody can be used to immunodeplete VACWR106, allowing assessment of its direct contribution to various steps in mRNA processing.

These approaches can provide insights into fundamental aspects of poxvirus gene expression and reveal potential targets for antiviral intervention.

What is the relationship between VACWR106 expression and the broader vaccinia virus transcriptional program?

VACWR106 plays a crucial role in the vaccinia virus transcriptional program. Vaccinia gene expression occurs in a cascade fashion with early, intermediate, and late phases. Research into poxvirus immunology has revealed that CD8+ T cells preferentially recognize early antigens, while CD4+ T cells target late and structural antigens .

The mRNA capping enzyme complex containing VACWR106 is particularly important for early gene expression. During this phase, the viral cores contain all necessary enzymes for transcription and mRNA processing, including VACWR106. Using the antibody in time-course experiments can reveal the dynamics of VACWR106 expression and localization throughout the viral life cycle.

Researchers can explore several questions about VACWR106 in the viral transcriptional program:

• How does inhibition of VACWR106 affect the expression of genes in different temporal classes?

• Does VACWR106 interact with different viral or host proteins during early versus late transcription?

• How does the subcellular localization of VACWR106 change throughout infection?

Understanding these relationships provides insights into the fundamental mechanisms of viral gene regulation and potential vulnerabilities that could be targeted therapeutically.

How can comparative studies using VACWR106 Antibody inform vaccine development efforts?

Comparative studies using VACWR106 Antibody can significantly contribute to vaccine development efforts by:

• Characterizing immune responses: The antibody can help assess whether VACWR106 is recognized by sera from vaccinated individuals or naturally infected hosts, providing insights into the immunogenicity of this viral component .

• Evaluating attenuated vaccine candidates: For potential new vaccinia-based vaccines, the antibody can help determine if mutations affect VACWR106 expression, localization, or function, which might impact viral replication and immunogenicity.

• Cross-protection studies: By examining cross-reactivity with orthologs from related poxviruses, researchers can assess the potential breadth of protection offered by vaccines targeting this protein.

• Correlates of protection: The antibody can be used to determine if recognition of VACWR106 correlates with protection in animal models, informing vaccine design.

Research has shown that effective protection against orthopoxviruses often requires antibodies targeting multiple viral components, with combinations of antibodies to both IMV and EEV providing superior protection . Understanding how VACWR106-specific antibodies contribute to this protection can inform the design of more effective vaccines or therapeutic antibody cocktails.

What are common challenges when using VACWR106 Antibody in immunoassays, and how can they be addressed?

Researchers may encounter several challenges when using VACWR106 Antibody:

For polyclonal antibodies like VACWR106 Antibody, lot-to-lot variation can be particularly challenging. Researchers should validate each new lot against a reference standard and consider creating a large stock of a well-performing lot for long-term studies.

How can researchers optimize antibody storage and handling to maintain VACWR106 Antibody performance?

Proper storage and handling are crucial for maintaining antibody performance:

• Temperature considerations: Store antibody aliquots at recommended temperatures (typically -20°C for long-term storage). Avoid repeated freeze-thaw cycles by preparing single-use aliquots. Working dilutions can usually be stored at 4°C for 1-2 weeks.

• Buffer composition: For diluted antibody preparations, consider adding stabilizing proteins (0.1-1% BSA) and preservatives (0.02% sodium azide) to prevent microbial growth and protein aggregation.

• Thermal stability assessment: Recent research on antibody fragments indicates melting temperatures (Tms) typically range from 50–70°C . While full antibodies generally have higher thermal stability, exposure to elevated temperatures should still be avoided.

• Centrifugation before use: Brief centrifugation (10,000 × g for 5 minutes) before use can remove protein aggregates that might cause non-specific binding.

• Documentation: Maintain detailed records of antibody performance across different experiments and lots to identify any degradation over time.

Researchers working with VACWR106 Antibody should also consider the stability of their viral antigen in experimental samples, as improper sample handling can lead to protein degradation and reduced antibody binding, independent of antibody quality.

What controls should be implemented when using VACWR106 Antibody in complex experimental systems?

Rigorous controls are essential for experiments using VACWR106 Antibody:

• Positive controls:

  • Lysates from cells infected with vaccinia virus Western Reserve strain

  • Recombinant VACWR106 protein (if available)

• Negative controls:

  • Uninfected cell lysates

  • Cells infected with a VACWR106 knockout or temperature-sensitive mutant virus (if available)

  • Primary antibody omission control

  • Isotype control (rabbit IgG at the same concentration)

• Specificity controls:

  • Peptide competition assay using the immunizing peptide/protein

  • Sequential probing of blots with antibodies to distinct viral proteins to confirm infection

• Application-specific controls:

  • For immunofluorescence: Include counterstains to verify cellular and subcellular context

  • For flow cytometry: Include fluorescence-minus-one (FMO) controls

  • For immunoprecipitation: Include a non-specific rabbit IgG control

• Quantification controls:

  • Standard curves using recombinant protein for quantitative applications

  • Loading controls (for Western blots) or normalization to cell number (for ELISAs)

Implementing these controls enables confident interpretation of results and helps troubleshoot any issues that may arise during experimental procedures.