TRPC5 Antibody

What is TRPC5 and why is it significant for research?

TRPC5 belongs to the transient receptor potential family and is one of the seven mammalian TRPC proteins. It functions as a multi-pass membrane protein that forms a receptor-activated non-selective calcium permeant cation channel. TRPC5 can function independently or as part of heteromultimeric assemblies with TRPC1, TRPC3, and TRPC4 . The significance of TRPC5 lies in its interactions with multiple proteins including calmodulin, CABP1, enkurin, Na(+)-H+ exchange regulatory factor (NHERF), interferon-induced GTP-binding protein (MX1), ring finger protein 24 (RNF24), and SEC14 domain and spectrin repeat-containing protein 1 (SESTD1) . Recent research has revealed its potential role in various pathological processes, particularly in the lung and neurological disorders, suggesting it could be a novel therapeutic target .

How do I select the appropriate TRPC5 antibody for my experiment?

Selection of the appropriate TRPC5 antibody depends on several experimental factors:

• Application requirements: Different antibodies perform optimally in specific applications. For instance, antibody 25890-1-AP has been validated for Western Blot (1:1000-1:4000 dilution), Immunohistochemistry (1:50-1:500 dilution), and Immunofluorescence (1:50-1:500 dilution) . The monoclonal antibody MA5-27657 has been specifically validated for detection of TrpC5 in rat brain lysate .

• Species reactivity: Confirm the antibody's reactivity with your experimental model. For example, 25890-1-AP shows reactivity with human and mouse samples .

• Antibody type: Consider whether a polyclonal or monoclonal antibody is more suitable. Polyclonal antibodies (like 25890-1-AP) recognize multiple epitopes, while monoclonal antibodies (like MA5-27657) target a single epitope, offering higher specificity but potentially lower sensitivity .

• Validation data: Review the antibody's validation data, including Western blot images showing the expected molecular weight (approximately 105-111 kDa for TRPC5) and tissue/cell line reactivity patterns .

Always perform preliminary validation experiments in your specific experimental system, as antibody performance can vary based on sample preparation methods and experimental conditions.

What are the optimal storage conditions for TRPC5 antibodies?

Proper storage is critical for maintaining antibody functionality and specificity. For TRPC5 antibodies, the following conditions are typically recommended:

Always refer to the manufacturer's specific recommendations for each antibody product, as storage requirements may vary slightly between different antibody preparations.

How can I optimize immunohistochemical detection of TRPC5 in tissue samples?

Optimizing immunohistochemical detection of TRPC5 requires careful attention to several methodological aspects:

• Antigen retrieval: For TRPC5 antibodies, heat-induced epitope retrieval is often essential. The 25890-1-AP antibody documentation suggests antigen retrieval with TE buffer pH 9.0, with an alternative option of citrate buffer pH 6.0 . The specific buffer should be empirically determined for different tissue types.

• Endogenous peroxidase blocking: To prevent false positive signals, endogenous peroxidases should be deactivated. Research on TRPC5 in lung tissues successfully employed treatment with 3% H₂O₂ solution to eliminate background .

• Antibody concentration: Titrate the antibody to determine optimal concentration. For IHC applications, a recommended starting dilution range for the 25890-1-AP antibody is 1:50-1:500 . Begin with manufacturer recommendations and adjust based on your specific tissue and fixation method.

• Controls: Include appropriate controls:

  • Positive controls: For TRPC5, human brain and cerebellum tissues have been validated as positive controls .

  • Negative controls: Omit primary antibody or use samples known to lack TRPC5 expression.

  • When possible, validate specificity using TRPC5-knockout tissues or TRPC5-knockdown samples .

• Signal development and detection: For chromogenic detection with DAB, ensure optimal conversion by the horseradish peroxidase of the antibody rather than endogenous peroxidases .

• Counterstaining: Use appropriate counterstaining (e.g., hematoxylin) that allows clear visualization of TRPC5 staining patterns while maintaining cellular morphological context.

By systematically optimizing these parameters, researchers can achieve specific and reproducible TRPC5 immunohistochemical staining across diverse tissue types.

What approaches can be used to validate TRPC5 antibody specificity?

Validating antibody specificity is critical for ensuring reliable experimental results. For TRPC5 antibodies, consider the following validation approaches:

• Genetic validation:

  • Use TRPC5-knockout models: The most definitive validation method is testing the antibody on tissues from TRPC5-knockout animals. Absence of signal in knockout samples strongly supports antibody specificity .

  • RNA interference: Knockdown TRPC5 using siRNA and confirm reduced antibody signal. This approach has been successfully employed in striatal cells to validate TRPC5 antibody specificity .

• Molecular weight verification:

  • Western blot analysis should show bands at the expected molecular weight. For TRPC5, the calculated molecular weight is 111 kDa, with observed molecular weight typically around 105-110 kDa .

  • Multiple band patterns may indicate splice variants, post-translational modifications, or degradation products.

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide before application to samples. Successful competition should eliminate or significantly reduce the specific signal.

• Orthogonal method comparison:

  • Compare protein detection with mRNA expression using RT-PCR or RNA-seq .

  • Use multiple antibodies targeting different epitopes of TRPC5.

• Cell/tissue expression patterns:

  • Verify that the staining pattern aligns with known TRPC5 expression profiles. For example, TRPC5 expression has been documented in specific lung structures and neuronal tissues .

When reporting results, always include detailed information about the validation methods used to support the specificity of the TRPC5 antibody employed in your research.

How can I detect TRPC5 S-glutathionylation in experimental models?

Detecting TRPC5 S-glutathionylation requires specialized techniques due to the transient nature of this post-translational modification. Based on research protocols, the following methodological approach is recommended:

• Protein expression system:

  • For low abundance TRPC5, consider transient expression of tagged TRPC5 (e.g., GFP-tagged TRPC5) to facilitate detection .

  • Be aware that overexpression may alter cellular responses to glutathionylation-inducing treatments.

• Glutathionylation induction:

  • Chemical induction using compounds like BCNU (carmustine) that increase cellular GSSG levels .

  • Direct application of GSSG (e.g., 5 mM) in patch-clamp experiments to activate TRPC5 currents .

  • Alternative induction using DTNP (2,2'-dithio-bis(5-nitropyridine)) to induce S-glutathionylation .

• Detection methods:

  • Immunoprecipitation of TRPC5 followed by Western blotting with anti-glutathione antibodies.

  • Electrophysiological measurements to detect TRPC5 channel activation as a functional readout of glutathionylation:

    • Whole-cell patch-clamp recording with GSSG in the pipette (5 mM) .

    • Characteristic TRPC5 current profiles: double-rectifying current for homomeric TRPC5 or outward rectifying current for TRPC1/C5 heteromeric channels .

• Validation approaches:

  • Pharmacological: Use selective TRPC5 antagonists like ML204 (10 μM) to confirm current identity .

  • Genetic: Compare responses in control cells versus TRPC5-knockdown cells treated with siTRPC5 .

  • Molecular: Verify channel expression and activation by constitutively active Gαi3 protein (Gαi3 Q204L) .

This methodology provides a comprehensive approach to investigating TRPC5 S-glutathionylation in experimental models relevant to neurodegenerative disorders and oxidative stress conditions.

How can TRPC5 antibodies be utilized to investigate its role in lung pathophysiology?

Recent research has revealed broader TRPC5 expression across lung tissues than previously recognized, suggesting important physiological and pathophysiological roles. To investigate TRPC5 in lung pathophysiology:

• Mapping cellular distribution:

  • Immunohistochemical analysis has successfully revealed TRPC5 expression in multiple lung structures including pleura, subpleural connective tissue, pulmonary arteries and veins, bronchioles, alveolar septa, type 1 and 2 pneumocytes, and alveolar macrophages .

  • Use anti-TRPC5 antibodies (e.g., 25890-1-AP at 1:50-1:500 dilution) with DAB visualization and appropriate controls .

• Quantitative expression analysis:

  • Western blot analysis of lung tissue lysates to quantify TRPC5 protein levels in healthy versus diseased states.

  • Combination with RT-PCR to correlate protein expression with mRNA levels in specific cell populations .

• Functional studies in isolated cells:

  • Isolate primary lung cells (e.g., airway smooth muscle cells, pulmonary arterial smooth muscle cells, alveolar macrophages) and examine TRPC5 function.

  • Use immunofluorescence to verify TRPC5 expression in cultured cells prior to functional experiments .

• Disease model investigations:

  • Compare TRPC5 expression and localization between healthy and pathological lung tissues.

  • Correlate TRPC5 expression patterns with clinical parameters and disease severity.

  • Investigate potential changes in TRPC5 distribution or expression in inflammatory lung diseases, fibrosis, or cancer.

• Therapeutic targeting assessment:

  • Use TRPC5 antibodies to evaluate the effects of potential therapeutic compounds on TRPC5 expression or localization.

  • Combine with functional assays to correlate changes in TRPC5 protein levels with altered cellular responses.

This comprehensive approach can provide insights into TRPC5's role in normal lung physiology and its potential contribution to pulmonary pathologies, possibly identifying new therapeutic approaches for lung diseases .

What methods can be used to study TRPC5's role in neurological disorders?

TRPC5 has been implicated in several neurological conditions, particularly those involving oxidative stress and calcium dysregulation. To investigate its role in neurological disorders:

• Electrophysiological characterization:

  • Whole-cell patch-clamp recordings to identify TRPC5-mediated currents in neuronal cells.

  • Distinctive current profiles help identify TRPC5: double-rectifying for homomeric TRPC5 versus predominantly outward rectifying for TRPC1/C5 heteromeric channels .

  • Validate using selective TRPC5 antagonist ML204 (10 μM) to confirm current identity .

• Molecular manipulation approaches:

  • RNA interference: Use siTRPC5 to knockdown TRPC5 expression in neuronal cell models (e.g., striatal cells) .

  • Overexpression studies: Express GFP-tagged TRPC5 to investigate effects of increased TRPC5 activity .

  • Constitutive activation: Use Gαi3 Q204L expression to augment TRPC5 currents .

• Oxidative stress models:

  • Induce oxidative stress with compounds like BCNU that increase GSSG levels and trigger TRPC5 S-glutathionylation .

  • Measure calcium influx using fluorescent indicators to assess TRPC5 activation.

  • Correlate TRPC5 activation with neuronal viability using apoptosis assays .

• Ex vivo tissue analysis:

  • Immunohistochemical staining of brain tissues from neurological disease models.

  • Quantify TRPC5 expression levels in affected versus unaffected brain regions.

  • Double-labeling with cell-type specific markers to identify neuronal populations expressing TRPC5.

• Therapeutic intervention assessment:

  • Test TRPC5 channel blockers (e.g., ML204) for neuroprotective effects in cellular models .

  • Measure outcomes including cell viability, apoptosis markers, and calcium homeostasis.

These methodological approaches have successfully demonstrated that inhibiting TRPC5 with ML204 prevents BCNU-induced cell death in striatal cells, suggesting TRPC5 as a potential therapeutic target in neurodegenerative conditions characterized by oxidative stress .

How do TRPC5 antibodies perform across different experimental systems?

TRPC5 antibody performance varies across experimental systems, requiring careful validation and optimization for each application. Based on available research:

*Specific antibody catalog numbers not provided in original research

Key considerations for optimal performance across systems:

• Tissue-specific optimization:

  • Antigen retrieval methods may differ between tissues: TE buffer pH 9.0 is recommended for brain tissue, with citrate buffer pH 6.0 as an alternative .

  • Endogenous peroxidase blocking is critical for lung tissue with high endogenous peroxidase activity .

• Species considerations:

  • Confirm reactivity with your species of interest. The 25890-1-AP antibody shows validated reactivity with human and mouse samples .

  • Cross-reactivity testing is recommended when using antibodies in non-validated species.

• Application-specific validation:

  • For electrophysiological studies, validate antibody specificity using siRNA knockdown and pharmacological inhibitors like ML204 .

  • For protein-protein interaction studies, validate that antibody binding doesn't interfere with the interaction interface.

• Signal amplification requirements:

  • Low-abundance TRPC5 in certain tissues may require signal amplification techniques or overexpression systems .

  • Consider GFP-tagging for visualization in systems with low endogenous expression .

This comparative analysis emphasizes the importance of antibody validation in each specific experimental context to ensure reliable and reproducible results when studying TRPC5 across different biological systems.

What are common technical challenges when using TRPC5 antibodies and how can they be addressed?

Researchers often encounter technical challenges when working with TRPC5 antibodies. Here are common issues and recommended solutions:

• Low signal intensity:

  • Cause: Insufficient antibody concentration, inadequate antigen retrieval, or low TRPC5 expression.

  • Solution: Increase antibody concentration (within manufacturer recommendations), optimize antigen retrieval protocols (try both TE buffer pH 9.0 and citrate buffer pH 6.0) , or use signal amplification systems like tyramide signal amplification.

  • Validation: Compare signals between known positive control tissues (e.g., brain tissue) and your experimental samples .

• High background/non-specific staining:

  • Cause: Insufficient blocking, cross-reactivity, or endogenous peroxidase activity.

  • Solution: Increase blocking time/concentration, pre-absorb antibody with non-specific proteins, or enhance endogenous peroxidase inactivation with 3% H₂O₂ treatment .

  • Validation: Include secondary-only controls to identify background from the detection system.

• Inconsistent Western blot results:

  • Cause: Protein degradation, incomplete transfer, or variable sample preparation.

  • Solution: Use fresh samples, add protease inhibitors, optimize transfer conditions for high molecular weight proteins (~105-111 kDa) .

  • Validation: Include positive control lysates from cells known to express TRPC5 (e.g., HL-60, MCF-7 cells) .

• Difficulties detecting endogenous TRPC5:

  • Cause: Low endogenous expression levels in many cell types.

  • Solution: Consider transient expression of tagged TRPC5, concentrate protein lysates, or use more sensitive detection methods .

  • Validation: Confirm antibody functionality using overexpression systems before attempting endogenous detection.

• Inability to detect functional changes:

  • Cause: Post-translational modifications affecting antibody binding or conformational changes.

  • Solution: Use multiple antibodies targeting different epitopes, complement with functional assays (e.g., calcium imaging, electrophysiology) .

  • Validation: Correlate protein detection with functional readouts like TRPC5 current in patch-clamp experiments .

By systematically addressing these technical challenges, researchers can significantly improve the reliability and reproducibility of their TRPC5 antibody-based experiments across various applications.

How can conflicting results between different TRPC5 antibodies be resolved?

Conflicting results between different TRPC5 antibodies are a common challenge in research. A systematic approach to resolving these discrepancies includes:

• Epitope mapping and antibody characterization:

  • Determine the exact epitopes recognized by each antibody. Differences in epitope accessibility due to protein conformation or post-translational modifications can cause discrepancies.

  • Verify whether antibodies target different isoforms or splice variants of TRPC5.

  • Compare polyclonal antibodies (which recognize multiple epitopes) with monoclonal antibodies (which target single epitopes) to understand potential differences .

• Comprehensive validation strategy:

  • Genetic validation: Test all antibodies on TRPC5-knockout/knockdown models .

  • Recombinant protein testing: Evaluate antibody binding to purified TRPC5 protein.

  • Peptide competition: Conduct competition assays with immunizing peptides for each antibody.

• Cross-platform verification:

  • Compare antibody performance across multiple detection methods:

    • Western blot: Evaluate band patterns and molecular weights (expected ~105-111 kDa) .

    • Immunohistochemistry/immunofluorescence: Compare staining patterns in established TRPC5-positive tissues .

    • Functional assays: Correlate antibody detection with electrophysiological measurements .

• Standardized experimental conditions:

  • Use identical sample preparation methods when comparing antibodies.

  • Apply consistent blocking and washing protocols.

  • Test antibodies at equivalent concentrations adjusted for their respective affinity.

• Independent verification:

  • Use orthogonal techniques that don't rely on antibodies:

    • mRNA detection (RT-PCR, RNA-seq) to confirm expression .

    • Mass spectrometry to validate protein identity.

    • CRISPR-tagged endogenous TRPC5 as a reference standard.

• Biological context consideration:

  • Evaluate whether conflicting results reflect actual biological variations rather than technical discrepancies:

    • Cell/tissue-specific post-translational modifications may affect epitope recognition.

    • Protein-protein interactions might mask certain epitopes in specific contexts.

By implementing this systematic troubleshooting approach, researchers can better understand the source of conflicting results and determine which antibody is most appropriate for their specific experimental context.

What emerging applications of TRPC5 antibodies show promise for therapeutic development?

TRPC5 antibodies are increasingly valuable tools for therapeutic research based on recent discoveries about TRPC5's role in various pathologies. Promising applications include:

• Respiratory disease therapeutics:

  • Recent findings demonstrate TRPC5 expression across all major lung structures, suggesting broader physiological roles than previously recognized .

  • TRPC5 antibodies enable screening of compounds that modulate TRPC5 function in airway smooth muscle cells, pulmonary arterial cells, and alveolar macrophages .

  • Quantifying changes in TRPC5 expression or localization in response to therapeutic candidates may identify novel approaches for treating lung diseases .

• Neurodegenerative disease interventions:

  • TRPC5 glutathionylation contributes to neuronal damage in conditions with oxidative stress, such as Huntington's disease .

  • Antibody-based screening assays can identify compounds that prevent TRPC5 S-glutathionylation or block activated channels.

  • ML204, a selective TRPC5 antagonist, prevents BCNU-induced cell death in striatal cells, demonstrating TRPC5 inhibition as a viable neuroprotective strategy .

• Target validation through immunoneutralization:

  • Function-blocking antibodies against extracellular domains of TRPC5 could serve as therapeutic prototypes.

  • Such antibodies could be used to validate TRPC5 as a drug target in animal models before developing small molecule inhibitors.

• Biomarker development:

  • TRPC5 antibodies facilitate studies correlating TRPC5 expression or modification levels with disease states.

  • Potential development of diagnostic assays measuring soluble TRPC5 or TRPC5-containing extracellular vesicles in patient samples.

• Structure-guided drug design:

  • Antibody co-crystallization studies with TRPC5 could reveal structural details to guide rational design of small molecule modulators.

  • Epitope mapping using various antibodies helps identify functional domains susceptible to pharmacological intervention.

These emerging applications leverage TRPC5 antibodies beyond basic research tools, positioning them as critical reagents for therapeutic development in respiratory, neurological, and potentially other disease areas where TRPC5 dysregulation contributes to pathophysiology.

How might novel TRPC5 antibody engineering approaches advance research capabilities?

Innovative antibody engineering approaches are poised to revolutionize TRPC5 research by overcoming current limitations and enabling new experimental paradigms:

• Conformation-specific antibodies:

  • Development of antibodies that selectively recognize active versus inactive TRPC5 conformations.

  • Applications: Real-time monitoring of channel activation status in living cells without electrophysiology.

  • Advantage: Ability to map spatial and temporal patterns of TRPC5 activation across tissues.

• Intrabodies for subcellular targeting:

  • Engineering cell-permeable antibody fragments that target TRPC5 intracellularly.

  • Applications: Selective inhibition of TRPC5 in specific subcellular compartments.

  • Advantage: Greater spatial resolution of TRPC5 function than possible with pharmacological agents.

• Bispecific antibodies:

  • Creating antibodies that simultaneously target TRPC5 and interacting proteins (calmodulin, CABP1, enkurin, NHERF, MX1, RNF24, or SESTD1) .

  • Applications: Investigation of specific protein-protein interactions in native contexts.

  • Advantage: Ability to selectively modulate specific TRPC5 signaling complexes.

• Modification-specific antibodies:

  • Developing antibodies that specifically recognize S-glutathionylated TRPC5 .

  • Applications: Direct detection of this post-translational modification without requiring electrophysiological measurements.

  • Advantage: Higher throughput screening of conditions affecting TRPC5 glutathionylation status.

• Nanobodies and single-domain antibodies:

  • Smaller antibody formats with enhanced tissue penetration and reduced immunogenicity.

  • Applications: Improved access to structurally restricted epitopes on TRPC5.

  • Advantage: Potentially superior for in vivo imaging or therapeutic applications.

• Antibody-fluorophore conjugates for super-resolution microscopy:

  • Direct conjugation of small fluorophores to TRPC5 antibodies.

  • Applications: Nanoscale visualization of TRPC5 distribution and co-localization.

  • Advantage: Detailed mapping of TRPC5 in multiprotein complexes at the membrane.

These innovative approaches extend beyond conventional antibody applications, promising to transform our understanding of TRPC5 biology through enhanced specificity, improved spatial and temporal resolution, and novel functional interrogation capabilities.

What are the most critical considerations for researchers working with TRPC5 antibodies?

Researchers working with TRPC5 antibodies must consider several critical factors to ensure reliable and reproducible results. The expanding role of TRPC5 across multiple physiological systems requires careful attention to experimental design and interpretation:

• Antibody validation is paramount: TRPC5 antibody specificity should be rigorously validated using genetic approaches (knockout/knockdown models), peptide competition assays, and multiple detection methods . This validation is particularly important given TRPC5's ability to form heteromeric channels with other TRPC family members, which may affect epitope accessibility .

• Context-dependent expression patterns: Recent research reveals TRPC5 expression in previously unrecognized tissues and cell types, such as various lung structures . Researchers should approach negative results cautiously and consider whether technical limitations rather than absence of expression may explain findings.

• Post-translational modifications affect detection: Modifications like S-glutathionylation can alter TRPC5 function and potentially antibody binding . Experimental conditions that induce oxidative stress may change TRPC5 detectability with certain antibodies.

• Functional correlation strengthens findings: Whenever possible, complement antibody-based detection with functional assays such as electrophysiology, calcium imaging, or cellular phenotypic responses to validate the physiological relevance of TRPC5 detection .

• Heteromeric channel complexes complicate interpretation: TRPC5 functions both independently and in heteromeric assemblies with TRPC1, TRPC3, and TRPC4 . This heterogeneity may affect antibody binding and experimental outcomes depending on the relative expression of different TRPC family members.