IRGC Antibody

What is IRGC and what is its biological significance?

IRGC (Interferon-inducible GTPase 5) belongs to the Immunity-related GTPases (IRG) family, also known as p47 GTPases. These proteins play critical roles in mediating innate resistance to intracellular pathogens. IRG genes have been identified in various mammals and lower species including mice, rats, zebrafish, and humans. In humans, only two IRG genes exist: IRGC and IRGM. Human IRGC encodes a full-length IRG protein that is constitutively expressed in testis, while IRGM encodes a considerably truncated protein expressed in cultured cells including some macrophage cell lines. Unlike mouse IRG genes, human IRG genes are not subject to interferon (IFN) control, suggesting that the host resistance mechanism supported by IRG proteins in mice may be absent in humans .

What are the key molecular characteristics of IRGC protein?

IRGC has several important molecular characteristics that researchers should note:

The discrepancy between calculated and observed molecular weights (50,288 Da vs. 68 kDa) may be attributed to post-translational modifications that affect protein migration in electrophoresis .

How do anti-IRGC antibodies function in experimental applications?

Anti-IRGC antibodies bind to specific epitopes on the IRGC protein, enabling its detection and visualization in various experimental applications. Most commercially available anti-IRGC antibodies are raised against synthetic peptides from specific regions of the protein. For example, one validated antibody was raised against a 12 amino acid synthetic peptide near the carboxy terminus of human IRGC, with the immunogen located within the last 50 amino acids of the protein. This strategic epitope selection enables recognition of both known IRGC isoforms .

The antibody-antigen interaction allows researchers to:

• Detect IRGC protein expression in tissue lysates using Western blot (WB)

• Visualize subcellular localization using immunocytochemistry (ICC)

• Examine cellular distribution patterns through immunofluorescence (IF)

• Quantify IRGC levels via enzyme-linked immunosorbent assay (ELISA)

What are the optimal conditions for Western blot analysis using anti-IRGC antibodies?

For effective Western blot analysis of IRGC, researchers should follow these validated methodological approaches:

• Antibody concentration: Use anti-IRGC antibody at a concentration of 1-2 μg/mL for optimal signal-to-noise ratio .

• Expected molecular weight: Look for a band at approximately 68 kDa, although the calculated molecular weight is 50,288 Da .

• Sample preparation: For validated results, mouse brain tissue lysate has been confirmed as an appropriate sample type .

• Controls: Include positive controls (tissues known to express IRGC) and negative controls (tissues with no IRGC expression or samples treated with blocking peptides) to validate specificity.

• Optimization: If signal strength varies, adjust antibody concentration within the recommended range and optimize exposure times accordingly.

Western blotting remains one of the most reliable methods for confirming IRGC protein expression and evaluating antibody specificity before proceeding to more complex applications .

How should researchers approach immunocytochemistry and immunofluorescence studies with IRGC antibodies?

For immunostaining applications, specific protocol adjustments are necessary to optimize IRGC detection:

For Immunocytochemistry (ICC):

• Use anti-IRGC antibody at 2.5 μg/mL concentration

• A20 cells have been validated as an appropriate cell model

• Include proper fixation and permeabilization steps to ensure antibody access to intracellular targets

• Use appropriate secondary antibodies matched to the host species (rabbit for the antibody described in the search results)

For Immunofluorescence (IF):

• Start with a higher concentration of 20 μg/mL of anti-IRGC antibody

• A20 cells have been validated for IF applications

• Use fluorophore-conjugated secondary antibodies appropriate for your imaging system

• Include DAPI or similar nuclear counterstain for localization context

For both techniques, researchers should optimize fixation methods, blocking solutions, and antibody incubation times based on their specific cell types and experimental conditions.

What storage and handling protocols ensure optimal IRGC antibody performance?

Proper storage and handling are critical for maintaining antibody integrity and experimental reproducibility:

• Storage temperature options:

  • Store at 4°C for up to three months for frequent use

  • Store at -20°C for up to one year for long-term storage

• Critical handling considerations:

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality

  • Do not expose antibodies to prolonged high temperatures

  • Prepare working dilutions fresh before use or store in small aliquots to prevent repeated freezing and thawing

• Working solution preparation:

  • Western blot: Dilute to 1-2 μg/mL

  • Immunocytochemistry: Dilute to 2.5 μg/mL

  • Immunofluorescence: Dilute to 20 μg/mL

• Buffer compatibility:

  • The antibody is typically supplied in PBS containing 0.02% sodium azide

  • Compatibility with common buffers (PBS, TBS) should be verified for specialized applications

Following these guidelines will maximize antibody performance and extend its functional lifespan across multiple experiments.

How can deep learning approaches enhance IRGC antibody development and optimization?

Recent advances in deep learning offer promising approaches for antibody development that could be applied to IRGC antibodies:

Wasserstein Generative Adversarial Networks with Gradient Penalty (WGAN+GP) models can generate novel antibody variable region sequences with desirable properties. While not specifically mentioned for IRGC antibodies, this approach could be adapted by:

• Creating training datasets: Collecting IRGC-binding antibody sequences with favorable properties like high specificity, affinity, and developability.

• Model training: Using these sequences to train a WGAN+GP model that learns the patterns associated with effective IRGC antibodies.

• In-silico screening: Computationally evaluating generated sequences for:

  • High percent humanness

  • Low chemical liabilities in complementarity-determining regions (CDRs)

  • High "medicine-likeness"

• Experimental validation: Testing promising candidates for:

  • Expression efficiency

  • Monomer content

  • Thermal stability

  • Low hydrophobicity and self-association

  • Minimal non-specific binding

This computational approach could significantly accelerate the development of high-quality IRGC antibodies while reducing the reliance on traditional antibody discovery methods that require animal immunization or display technologies .

How should researchers validate IRGC antibody specificity?

Rigorous validation is essential for ensuring experimental reliability and reproducibility. A comprehensive validation approach includes:

• Multiple application testing: Confirm consistent results across different applications (WB, ICC, IF, ELISA) as demonstrated in the available validation data .

• Blocking peptide experiments: Use the immunizing peptide (the 12 amino acid synthetic peptide from the carboxy terminus of human IRGC) to competitively inhibit antibody binding in control experiments .

• Genetic validation: Test antibody reactivity in IRGC-knockout or knockdown samples, which should show significantly reduced or absent signal.

• Isoform recognition: Verify that the antibody recognizes both known isoforms of IRGC as claimed in product specifications .

• Cross-species validation: Confirm reactivity in each claimed species (Human, Mouse, Rat) using appropriate positive control samples .

• Testing across multiple tissue/cell types: Evaluate antibody performance in various IRGC-expressing tissues beyond those in validation data.

• Cross-reactivity assessment: Examine potential cross-reactivity with other related GTPases, particularly other IRG family members.

What strategies help troubleshoot unexpected molecular weight variations in IRGC detection?

The discrepancy between calculated (50,288 Da) and observed (68 kDa) molecular weights of IRGC requires careful interpretation:

• Post-translational modifications: Consider potential glycosylation, phosphorylation, or other modifications that could increase apparent molecular weight.

• Isoform detection: The antibody may be detecting specific IRGC isoforms with different molecular weights than predicted from the primary sequence.

• Detergent and reducing agent effects: Experiment with different sample preparation conditions, including various detergents and reducing agent concentrations.

• Gel percentage optimization: Use gradient gels or different acrylamide percentages to improve resolution in the molecular weight range of interest.

• Confirm antibody specificity: Use additional validation methods (peptide blocking, knockout controls) to verify that the observed band is indeed IRGC.

• Literature comparison: Compare your findings with published literature reporting IRGC molecular weight variations to identify consistent patterns .

Understanding these variations is crucial for accurate data interpretation and can provide insights into the post-translational regulation of IRGC in different biological contexts.

How does IRGC expression and function compare between human and mouse models?

Understanding species differences is crucial for translational research:

These differences suggest that mouse models may not fully recapitulate human IRGC biology, necessitating careful experimental design when studying IRGC across species. The divergent regulation and expression patterns indicate potentially different evolutionary trajectories for this immune component between species .

How can IRGC antibodies contribute to seroprevalence studies similar to COVID-19 research?

While the search results don't directly link IRGC antibodies to seroprevalence studies, antibody-based detection methodologies from COVID-19 research provide insights for other applications:

• Assay development principles: The ELISA-based detection of SARS-CoV-2-specific IgG and IgM antibodies in population studies demonstrates how similar approaches could be developed for detecting anti-IRGC antibodies in research or clinical contexts .

• Methodological considerations:

  • Population sampling strategies (as described in the Iranian seroprevalence study) provide models for collecting representative data

  • Adjustment for test performance characteristics (sensitivity and specificity) is critical for accurate interpretation

  • Multiple testing timepoints may be necessary to track antibody development and persistence

• Special populations analysis: The multicenter study of antibody seroprevalence in cancer patients offers a template for investigating IRGC in specific patient populations of interest .

• Data interpretation frameworks: The approaches for estimating previous infection rates from antibody data in COVID-19 studies could inform analysis of other antibody-based population studies .

These methodologies could be adapted for investigating IRGC expression patterns in population studies or clinical research settings if IRGC becomes a biomarker of interest for specific conditions.

What novel approaches could enhance IRGC antibody sensitivity and specificity?

Several emerging technologies could improve IRGC antibody performance:

• Antibody engineering approaches:

  • Using deep learning models like WGAN+GP to design optimized antibody sequences

  • Employing structure-based design to target specific IRGC epitopes

  • Developing recombinant antibody fragments (Fab, scFv) for improved tissue penetration

• Detection enhancement strategies:

  • Signal amplification methods such as tyramide signal amplification for immunohistochemistry

  • Proximity ligation assays for detecting IRGC interactions with binding partners

  • Super-resolution microscopy techniques for detailed subcellular localization studies

• Validation technologies:

  • CRISPR-Cas9 generated IRGC knockout cell lines as definitive negative controls

  • Orthogonal validation with mass spectrometry-based proteomics

  • Multiplexed detection systems to simultaneously visualize IRGC and interaction partners

These approaches would build upon the foundation of current IRGC antibody technology to expand research capabilities and enhance data reliability .

How might IRGC antibodies contribute to understanding tissue-specific immune mechanisms?

Given IRGC's constitutive expression in testis, antibody-based studies could reveal important tissue-specific immune functions:

• Reproductive immunology: Investigating IRGC's role in testis immune privilege or response to reproductive tract infections.

• Comparative immunology: Using anti-IRGC antibodies to map expression patterns across species, potentially revealing evolutionary adaptations in tissue-specific immunity.

• Signaling pathway analysis: Employing co-immunoprecipitation with anti-IRGC antibodies to identify tissue-specific binding partners and signaling cascades.

• Pathogen response mechanisms: Exploring whether human IRGC contributes to resistance against specific testis-tropic pathogens despite its different regulation compared to mouse IRGs.

• Developmental immunology: Tracking IRGC expression during development to understand its regulation and potential role in establishing tissue-specific immune environments.

These research directions would leverage anti-IRGC antibodies as tools to uncover the specialized functions of this GTPase in human physiology and pathology .