TCIRG1 Antibody

What is TCIRG1 and what are its main biological functions?

TCIRG1 functions as a critical component of vacuolar ATPase (V-ATPase), specifically as the V0 subunit A3. This protein is essential for cellular acidification processes that support metabolism, membrane transport, and intracellular signaling pathways. Research has revealed two main isoforms: TCIRG1-isoa (the longer variant) and TCIRG1-isob (which lacks the first 5 exons of isoa). The protein is expressed in multiple tissues including heart, liver, kidney, lung, and pancreas, with particularly notable roles in immune regulation and bone resorption. Recent studies have also identified TCIRG1's involvement in congenital neutropenia and its potential role as a prognostic biomarker in certain cancers, most notably clear cell renal cell carcinoma (ccRCC), where it appears to influence tumor progression .

How do I select the appropriate TCIRG1 antibody for my specific research application?

Selecting the optimal TCIRG1 antibody requires careful consideration of several factors:

• Target epitope: Different antibodies target distinct regions of TCIRG1. For example, some antibodies recognize the N-terminal cytoplasmic domain (AA 1-130), while others target regions AA 121-220 or other segments. Choose an antibody that targets the region most relevant to your research question and accessible in your experimental conditions .

• Isoform specificity: Consider whether your research requires detection of specific TCIRG1 isoforms. Some antibodies detect both TCIRG1-isoa (~45kDa) and TCIRG1-isob, while others are isoform-specific .

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IF, ELISA, FACS). For example, antibody ABIN7270707 is validated for WB, IF, and IHC applications .

• Host species: Consider the host species (rabbit, mouse) in relation to your experimental design, especially for multi-color immunostaining where avoiding cross-reactivity is essential .

• Clonality: Polyclonal antibodies offer broader epitope recognition but potential batch variation, while monoclonal antibodies provide higher specificity and reproducibility for quantitative analyses .

What are the validated cross-reactivity profiles of common TCIRG1 antibodies?

Most commercial TCIRG1 antibodies have been validated for human samples, with varying cross-reactivity to mouse and rat TCIRG1. For instance, the polyclonal antibody ABIN7270707 (targeting AA 1-130) demonstrates cross-reactivity with human, mouse, and rat samples, making it suitable for comparative studies across these species. When selecting antibodies for cross-species research, prioritize those with explicitly validated cross-reactivity rather than relying on sequence homology predictions. The antibody's validated applications may also differ between species - some antibodies work for Western blotting in human samples but only for immunofluorescence in mouse/rat samples. Always review the validation data specific to your species and application of interest .

What are the optimal protein extraction methods for Western blot detection of TCIRG1?

For effective TCIRG1 Western blot analysis, the protein extraction method must preserve the membrane-associated properties of this V-ATPase subunit while minimizing protein degradation:

• Buffer selection: Use RIPA buffer supplemented with protease inhibitors for general applications. For enhanced membrane protein extraction, consider using buffers containing 1-2% NP-40 or Triton X-100.

• Sample preparation: For peripheral blood mononuclear cells (PBMCs), which have been successfully used in TCIRG1 studies, use RBC lysis followed by gentle membrane disruption to maintain protein integrity .

• Protein quantification: Due to TCIRG1's varying expression levels across tissues, standardize loading using housekeeping proteins that are stably expressed in your experimental context.

• Denaturation conditions: Heat samples at 70°C (not 95°C) for 10 minutes in Laemmli buffer containing 2-5% β-mercaptoethanol to reduce aggregation of this membrane protein.

• Gel selection: Use 10-12% polyacrylamide gels for optimal resolution of TCIRG1's ~45kDa fragment and other isoforms .

When analyzing Western blot results, note that different antibodies may detect distinct fragments of TCIRG1. For instance, polyclonal antibodies against the N-terminal domain detect the ~45kDa TCIRG1 fragment (likely TCIRG1-isob), while other antibodies may detect different isoforms or processed variants .

How can I optimize immunohistochemistry protocols for TCIRG1 detection in different tissue types?

Optimizing IHC protocols for TCIRG1 requires adjustments based on tissue type and fixation method:

• Antigen retrieval: For formalin-fixed paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval with citrate buffer (pH 6.0) typically yields optimal results for TCIRG1 antibodies. For tissues with high extracellular matrix content, consider extending retrieval time.

• Antibody concentration: Titrate antibody concentrations beginning with the manufacturer's recommended dilution. TCIRG1 antibodies typically require optimization between 1:100-1:500 dilutions depending on tissue type and fixation conditions .

• Incubation conditions: For stronger signals in tissues with low TCIRG1 expression, extend primary antibody incubation to overnight at 4°C rather than standard 1-hour incubations.

• Detection system: For tissues with low expression levels, amplification systems like tyramide signal amplification (TSA) or polymer-based detection may enhance sensitivity while maintaining specificity.

• Validation controls: Include positive controls (such as renal cell carcinoma tissues for TCIRG1 overexpression) and negative controls (antibody diluent only) to validate staining specificity .

For multi-color IHC detecting TCIRG1 alongside other markers, sequential staining protocols may be necessary to avoid antibody cross-reactivity, particularly when studying TCIRG1 in immune cell populations.

What are the recommended cell models for studying TCIRG1 function in vitro?

Based on the available research data, several cell models have been validated for TCIRG1 functional studies:

When selecting cell models, consider the specific TCIRG1 isoform expression patterns in different cell types. For functional studies involving TCIRG1 knockdown, ccRCC cell lines have been successfully used to demonstrate the role of TCIRG1 in cancer cell migration . For studies focused on TCIRG1's role in immune regulation, primary immune cells or appropriate immune cell lines should be selected based on the specific immune cell population of interest.

How can I address non-specific binding issues when using TCIRG1 antibodies?

Non-specific binding is a common challenge with TCIRG1 antibodies, particularly in applications like Western blotting and immunohistochemistry. To address this issue:

• Blocking optimization: Extended blocking (2 hours at room temperature) with 5% non-fat milk in TBST for Western blots, or 10% normal serum from the same species as the secondary antibody for IHC/IF, can significantly reduce non-specific binding.

• Antibody dilution: Titrate your antibody carefully, as both insufficient and excessive concentrations can contribute to non-specific binding. Start with the manufacturer's recommended dilution and adjust based on signal-to-noise ratio .

• Washing stringency: Increase the stringency of washing steps by adding 0.1-0.3% Tween-20 to washing buffers and extending washing durations. For particularly problematic samples, consider including a high-salt (500mM NaCl) wash step.

• Alternative antibodies: If persistent non-specific binding occurs, compare results using antibodies targeting different epitopes of TCIRG1. For example, if an antibody against the N-terminal domain (AA 1-130) shows non-specific binding, try an antibody targeting AA 121-220 .

• Validation controls: Always include appropriate negative controls, such as tissues or cells known to lack TCIRG1 expression, or ideally, TCIRG1 knockout/knockdown samples, to identify true non-specific binding patterns.

How should I interpret discrepancies in TCIRG1 detection between different antibodies or techniques?

Discrepancies in TCIRG1 detection are often attributable to several factors that require careful interpretation:

• Isoform specificity: TCIRG1 has multiple isoforms, including TCIRG1-isoa and TCIRG1-isob, which lack the first 5 exons of the longer variant. Different antibodies may preferentially detect specific isoforms, leading to apparent discrepancies in expression levels .

• Epitope accessibility: The conformation of TCIRG1 differs between applications - denatured in Western blots versus native in immunoprecipitation or flow cytometry. Antibodies targeting conformational epitopes may show different results across techniques.

• Post-translational modifications: Modifications may mask epitopes in certain contexts. For example, studies have shown detection of reduced amounts of ~45kDa TCIRG1 product fragments in individuals with mutations, suggesting possible altered processing or stability .

• Technical sensitivity: Western blotting typically offers higher sensitivity for low-abundance proteins compared to IHC. When discrepancies occur, quantitative PCR for TCIRG1 mRNA can provide complementary data to resolve conflicts.

When faced with discrepancies, employ multiple antibodies targeting different epitopes and use complementary techniques (e.g., RNA-seq or qPCR) to validate protein expression findings. Document the specific antibody used, detection method, and sample preparation for accurate interpretation of results across studies.

What considerations should I take into account when using TCIRG1 antibodies for co-localization studies?

Co-localization studies involving TCIRG1 require specific considerations due to its subcellular distribution and multiple isoforms:

• Fixation effects: TCIRG1's membrane association makes it sensitive to fixation artifacts. Compare paraformaldehyde fixation (2-4%) with methanol fixation to determine optimal epitope preservation while maintaining cellular architecture.

• Permeabilization optimization: As a membrane protein component of V-ATPase, TCIRG1 requires balanced permeabilization - sufficient for antibody access but not excessive to disrupt membrane structures. Try 0.1% Triton X-100 or 0.05% saponin, adjusting based on signal quality.

• Sequential staining: For co-localization with other markers, especially when antibodies are raised in the same species, sequential staining protocols with appropriate blocking steps between primary antibodies are recommended.

• Spectral considerations: Choose fluorophores with minimal spectral overlap when designing multi-color immunofluorescence experiments, particularly important when co-localizing TCIRG1 with other V-ATPase components.

• Super-resolution techniques: Consider super-resolution microscopy techniques (STED, STORM, SIM) for precise subcellular localization of TCIRG1, especially when studying its distribution relative to endosomal/lysosomal markers.

When interpreting co-localization results, quantify overlap using appropriate coefficients (Pearson's, Mander's) rather than relying on visual assessment alone. This provides more objective assessment of TCIRG1's spatial relationship with other proteins of interest.

How can TCIRG1 antibodies be utilized to study its role in congenital neutropenia?

Investigating TCIRG1's role in congenital neutropenia requires specialized approaches using antibodies:

• Genetic-protein correlation: Use Western blot analysis with TCIRG1 antibodies to assess protein expression in neutropenic patients with known TCIRG1 mutations. Previous studies have shown reduced levels of ~45kDa TCIRG1 product fragment in affected individuals carrying mutations like ARG736SER, compared to healthy controls .

• Isoform-specific detection: Employ antibodies with known isoform specificity to distinguish between TCIRG1-isoa and TCIRG1-isob expression, as mutations may differentially affect these isoforms. A polyclonal antibody developed against the N-terminal cytoplasmic domain can detect the shorter TCIRG1-isob, while monoclonal antibodies against partial recombinant TCIRG1 isoa 121aa-220aa detect distinct patterns .

• Functional assays: Combine antibody-based detection with functional assays measuring neutrophil development and survival in patient samples. Correlation between TCIRG1 protein levels and functional outcomes can provide mechanistic insights.

• Tissue distribution analysis: Use immunohistochemistry with validated TCIRG1 antibodies to compare TCIRG1 distribution in bone marrow samples from patients versus controls, focusing on granulocyte precursor populations.

• Protein interaction studies: Employ co-immunoprecipitation with TCIRG1 antibodies to identify altered protein interactions in neutropenic patients that might explain the link between TCIRG1 mutations and neutrophil development.

This comprehensive approach can help elucidate how TCIRG1 mutations contribute to the pathogenesis of congenital neutropenia, potentially revealing novel therapeutic targets.

What methodologies are recommended for studying TCIRG1's role in tumor immunity and cancer progression?

Research into TCIRG1's oncogenic functions can be approached through several antibody-dependent methodologies:

• Expression profiling: Use validated TCIRG1 antibodies for IHC analysis of tissue microarrays to correlate TCIRG1 expression with clinical outcomes across cancer types. TCIRG1 overexpression has been documented in several cancers, particularly ccRCC, where it predicts unfavorable clinical outcomes .

• Immune infiltrate characterization: Combine TCIRG1 IHC with immune cell markers to analyze relationships between TCIRG1 expression and tumor-infiltrating lymphocytes. TCIRG1 has shown strong associations with CD8+ T-cell, Treg, and CD4+ T-cell infiltration patterns in tumors .

• Cell line models: Implement TCIRG1 knockdown/overexpression in cancer cell lines followed by Western blot confirmation of altered expression, enabling functional studies of migration and invasion. Experimental evidence indicates that TCIRG1 knockdown inhibits migration of ccRCC cells .

• Therapeutic response prediction: Correlate TCIRG1 expression levels (detected by IHC or Western blot) with response to immunotherapy treatments. TCIRG1 levels have been associated with Tumor Immune Dysfunction and Exclusion (TIDE) scores, suggesting potential value in predicting immunotherapy response .

• Mechanistic pathway analysis: Use phospho-specific antibodies alongside TCIRG1 antibodies to investigate downstream signaling pathways activated in TCIRG1-overexpressing tumors, potentially revealing therapeutic targets.

This multi-faceted approach allows for comprehensive investigation of TCIRG1's roles in both tumor progression and immune evasion mechanisms.

How can I design effective validation strategies for TCIRG1 antibody specificity?

Rigorous validation of TCIRG1 antibody specificity is essential for confident data interpretation. A comprehensive validation strategy should include:

• Genetic knockout/knockdown controls: The gold standard for antibody validation involves testing the antibody in TCIRG1 knockout or knockdown models. Compare Western blot or IHC signals between wild-type and TCIRG1-depleted samples to confirm specificity.

• Peptide competition assays: Pre-incubate the TCIRG1 antibody with excess immunizing peptide before application to samples. Specific binding should be competitively inhibited, while non-specific binding will persist.

• Multiple antibody concordance: Test at least two antibodies targeting different epitopes of TCIRG1 (e.g., N-terminal domain versus middle region) and compare detection patterns. Consistent results across different antibodies increase confidence in specificity .

• Cross-species reactivity assessment: If the antibody claims cross-reactivity with multiple species, test samples from each species to confirm consistent detection of the appropriately sized protein band or expected localization pattern .

• Correlation with orthogonal methods: Compare protein detection results with mRNA expression data from qPCR or RNA-seq to confirm that protein and transcript levels correlate in the experimental system.

• Mass spectrometry validation: For ultimate confirmation, perform immunoprecipitation with the TCIRG1 antibody followed by mass spectrometry analysis to verify the identity of the captured proteins.

Document these validation steps thoroughly when publishing research using TCIRG1 antibodies, as this improves reproducibility and confidence in the findings.

What methodological approaches are recommended for studying TCIRG1 isoform-specific functions?

Investigating isoform-specific functions of TCIRG1 requires specialized methodological approaches:

• Isoform-specific detection: Select antibodies with validated specificity for either TCIRG1-isoa or TCIRG1-isob. For instance, antibodies targeting the first 5 exons will detect only the longer TCIRG1-isoa, while those targeting shared regions will detect both isoforms but require careful band size interpretation in Western blots .

• Isoform-selective knockdown: Design siRNA or shRNA constructs targeting unique regions of each isoform, followed by validation of isoform-specific reduction using appropriate antibodies. This allows for functional studies distinguishing the roles of different TCIRG1 variants.

• Expression systems: For overexpression studies, construct expression vectors containing the specific TCIRG1 isoform of interest, and confirm expression using isoform-specific antibodies before proceeding to functional analyses.

• Tissue distribution mapping: Use isoform-specific antibodies to map the differential distribution of TCIRG1 isoforms across tissues, particularly focusing on hematopoietic and immune cell populations relevant to neutropenia and immune regulation .

• Co-immunoprecipitation analyses: Employ isoform-specific antibodies for co-immunoprecipitation studies to identify potential isoform-specific protein interaction partners that might explain their distinct biological functions.

These approaches allow for precise delineation of the distinctive roles of TCIRG1 isoforms in cellular processes, disease pathogenesis, and potential therapeutic targeting.

How can I integrate TCIRG1 antibody-based techniques with genomic and transcriptomic analyses for comprehensive studies?

Multi-omics integration with TCIRG1 antibody-based techniques enables deeper mechanistic insights:

• Genotype-phenotype correlations: Pair TCIRG1 antibody detection (by Western blot or IHC) with genomic sequencing data to correlate specific TCIRG1 mutations or variants with protein expression levels and localization patterns. This approach has successfully identified association between TCIRG1 mutations and congenital neutropenia .

• Transcriptome-proteome integration: Correlate TCIRG1 mRNA expression from RNA-seq or microarray data with protein levels detected by TCIRG1 antibodies to identify potential post-transcriptional regulatory mechanisms or protein stability factors.

• Epigenetic regulation: Combine chromatin immunoprecipitation (ChIP) data on TCIRG1 promoter regions with antibody-based TCIRG1 protein detection to investigate epigenetic mechanisms controlling TCIRG1 expression. Studies have shown relationships between TCIRG1 expression and DNA methylation status .

• Pathway analysis validation: Use antibody-based techniques to validate key protein interactions or modifications predicted from computational analyses of transcriptomic data, strengthening the biological relevance of in silico findings.

• Single-cell integration: Pair single-cell RNA-seq data with single-cell protein detection methods (such as mass cytometry or imaging mass cytometry using TCIRG1 antibodies) to resolve cell-type specific expression patterns at both RNA and protein levels.

This integrated approach has been productive in cancer research, where TCIRG1 antibody-based detection combined with genomic analyses has revealed associations with cancer stemness indicators and somatic mutation patterns (particularly PBRM1 and BAP1 mutations in renal cancer) .

What considerations should be taken into account when developing TCIRG1-targeted therapeutic approaches?

Developing therapeutic strategies targeting TCIRG1 requires careful antibody-based validation steps:

• Expression profiling: Use validated TCIRG1 antibodies to comprehensively map expression across healthy tissues to predict potential on-target toxicities of TCIRG1-directed therapies. Note TCIRG1's expression in heart, liver, kidney, lung, and pancreas .

• Function validation: Before therapeutic targeting, confirm the causal role of TCIRG1 in disease pathogenesis through antibody-based detection following genetic manipulation in relevant model systems. Experimental evidence shows that TCIRG1 knockdown inhibits migration of cancer cells, supporting its potential as a therapeutic target .

• Biomarker development: Develop reliable IHC protocols with TCIRG1 antibodies for potential use as companion diagnostics to identify patients likely to benefit from TCIRG1-targeted therapies, particularly in cancers where TCIRG1 overexpression correlates with poor outcomes .

• Isoform selectivity: Determine whether therapeutic approaches should target all TCIRG1 isoforms or specific variants based on their differential expression in disease states. This requires isoform-specific antibodies for validation studies .

• Immune impact assessment: Given TCIRG1's association with immune cell infiltration in tumors, use multiparameter immunofluorescence with TCIRG1 and immune cell marker antibodies to predict how TCIRG1-targeted therapies might alter tumor immunology .