TRPC1 Antibody

What is TRPC1 and why is it important in scientific research?

TRPC1 is a member of the Transient Receptor Potential (TRP) superfamily, which represents one of the largest ion channel families. It was the first mammalian TRP protein reported to form an ion channel . The protein is approximately 91.2 kilodaltons in mass and consists of 793 amino acids . TRPC1's significance in research stems from its crucial role in store-operated calcium entry (SOCE), which regulates calcium homeostasis in cells . This process is fundamental to multiple physiological functions including muscle contraction, neurotransmitter release, and various cellular signaling pathways . TRPC1 can also co-assemble with other TRPC subunits (TRPC3, TRPC4, TRPC5) to form heteromeric channels with distinct properties, adding complexity to calcium signaling research .

How does TRPC1 relate to other TRP channel subfamilies?

The mammalian TRP superfamily comprises six subfamilies:

• TRPC (canonical)

• TRPV (vanilloid)

• TRPM (melastatin)

• TRPML (mucolipins)

• TRPP (polycystin)

• TRPA (ANKTM1)

The TRPC subfamily itself consists of seven proteins (TRPC1-7) further divided into four subgroups based on sequence homology and functional similarities:

• TRPC1

• TRPC4 and TRPC5

• TRPC3, TRPC6, TRPC7

• TRPC2

TRPC1 stands apart as its own subgroup due to its unique structural and functional characteristics. While all TRP channels are involved in calcium signaling, TRPC1's specific role in store-operated calcium entry distinguishes it from other TRP channels that might respond primarily to other stimuli such as temperature, mechanical force, or chemical ligands.

What are the common alternative names and identifiers for TRPC1?

When searching literature or databases for TRPC1, researchers should be aware of several alternative nomenclature designations:

• HTRP-1

• TRP1

• Short transient receptor potential channel 1

• Capacitative calcium channel protein Trp1

The human TRPC1 protein has the accession number P48995 in protein databases . When working with experimental models, it's important to note that TRPC1 has orthologs in multiple species including canine, porcine, monkey, mouse, and rat models, allowing for comparative studies across species .

What are the primary applications for TRPC1 antibodies in research?

TRPC1 antibodies serve multiple experimental purposes depending on the antibody type and research objective:

Each application requires specific optimization of antibody dilution, incubation conditions, and detection methods to achieve reliable results.

How should researchers validate the specificity of TRPC1 antibodies?

Antibody validation is a critical step to ensure experimental reliability. For TRPC1 antibodies, a comprehensive validation approach includes:

• Knock-out (KO) validation: Test the antibody in tissues or cells where TRPC1 has been genetically deleted. Absence of signal in KO samples confirms specificity .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (e.g., peptide QLYDKGYTSKEQKDC for ACC-010 antibody) before application. Specific binding should be blocked, as demonstrated in western blot analysis of rat brain membranes .

• Multiple antibody approach: Use antibodies targeting different epitopes of TRPC1 and compare the detection patterns.

• Cross-reactivity assessment: Test the antibody against closely related proteins (other TRPC family members) to ensure it doesn't cross-react.

• Multi-technique verification: Confirm protein expression using complementary techniques (e.g., if detected by WB, verify with IF or IHC).

The search results specifically mention a KO-validated Anti-TRPC1 Antibody (ACC-010), which provides higher confidence in antibody specificity .

What optimization strategies should be employed for Western blot detection of TRPC1?

Western blot detection of TRPC1 requires specific optimization due to its membrane protein nature and potential for post-translational modifications:

• Sample preparation:

  • Use specialized membrane protein extraction buffers containing appropriate detergents

  • Avoid excessive heating which may cause aggregation of membrane proteins

  • Include protease inhibitors to prevent degradation

• Gel electrophoresis:

  • Use gradient gels (e.g., 4-12%) to effectively resolve the ~91.2 kDa TRPC1 protein

  • Consider native PAGE for studying TRPC1 in complexes with other proteins

• Transfer conditions:

  • Employ wet transfer methods for more efficient transfer of larger proteins

  • Use lower methanol concentrations to improve transfer of hydrophobic proteins

• Antibody incubation:

  • Optimize antibody dilutions (typically 1:200 for Anti-TRPC1 as seen in rat brain membrane studies)

  • Extended incubation times at 4°C may improve signal-to-noise ratio

  • Include appropriate blocking agents to reduce background

• Controls:

  • Always include positive controls (tissues known to express TRPC1 such as brain)

  • Consider using recombinant TRPC1 as a size reference

  • Include peptide competition controls to verify specificity

These optimization steps are crucial for obtaining reliable and reproducible Western blot results for TRPC1 detection.

What is the tissue distribution pattern of TRPC1 in mammals?

TRPC1 exhibits a distinct expression pattern across mammalian tissues:

• High expression:

  • Central nervous system (particularly in the brain)

  • Fetal and adult brain (including cerebellum)

  • Adult heart

  • Reproductive tissues (testis and ovary)

• Moderate expression:

  • Endothelial cells (demonstrated in Human Umbilical Vein Endothelial Cells - HUVECs)

  • Bovine aortic endothelium (BAE-1) cells

• Cell type-specific expression in brain:

  • Purkinje cells in cerebellum

  • Molecular and granule layers of cerebellum

  • Human U373 MG cells (glial cell line)

This tissue distribution profile provides important context for experimental design, suggesting tissue sources for positive controls and highlighting physiologically relevant systems for functional studies.

How can immunohistochemistry be optimized for TRPC1 detection in tissue sections?

Optimizing immunohistochemistry (IHC) for TRPC1 detection requires careful attention to several factors:

• Tissue preparation:

  • For TRPC1 detection in brain tissues, frozen sections have been successfully used rather than paraffin-embedded tissues

  • Consider antigen retrieval methods for formalin-fixed tissues to expose epitopes

  • Test both perfusion fixation and immersion fixation protocols

• Antibody selection and dilution:

  • Anti-TRPC1 Antibody (ACC-010) has been validated for IHC in mouse cerebellum at specific dilutions

  • Dilution ratios between 1:80 (as used in U373 MG cells) and 1:200 (as used in BAE-1 cells) may serve as starting points

  • Both monoclonal (e.g., E-6) and polyclonal antibodies can be effective for IHC applications

• Detection systems:

  • Fluorescent secondary antibodies allow for co-localization studies with other markers

  • DAB-based chromogenic detection provides permanent specimens for long-term archiving

• Co-localization markers:

  • Parvalbumin has been used successfully as a marker for co-localization studies with TRPC1 in cerebellar Purkinje cells

  • Consider cell-type specific markers depending on the tissue under investigation

• Controls:

  • Include peptide competition controls

  • Use tissues from TRPC1 knockout animals as negative controls when available

Following these optimization steps enables reliable visualization of TRPC1 distribution patterns in tissue sections and cellular compartments.

How does TRPC1 contribute to store-operated calcium entry (SOCE)?

TRPC1 plays a fundamental role in store-operated calcium entry, a process triggered by depletion of intracellular calcium stores:

• Mechanism of action:

  • TRPC1 contributes to the formation of capacitative calcium entry complexes

  • The interaction between inositol 1,4,5-trisphosphate receptors and TRPC1 is essential for the activation of store-mediated calcium entry

  • TRPC1 functions as a component of calcium-permeable channels that activate in response to depletion of intracellular calcium stores

• Functional partners:

  • TRPC1 can form heteromeric channels with other TRPC subunits (TRPC3, TRPC4, TRPC5)

  • It interacts with key calcium signaling proteins including STIM1 (stromal interaction molecule 1) and Orai1

  • These protein-protein interactions can be studied using co-immunoprecipitation techniques with TRPC1 antibodies

• Physiological significance:

  • SOCE is vital for various calcium-dependent processes including:

    • Muscle contraction

    • Neurotransmitter release

    • Cellular signaling cascades

  • Dysregulation of TRPC1-mediated SOCE has been implicated in various pathological conditions

Understanding these molecular interactions has significant implications for research into calcium signaling disorders and potential therapeutic interventions.

What experimental approaches can distinguish between TRPC1 homomeric and heteromeric channels?

Distinguishing between TRPC1 homomeric channels and heteromeric assemblies with other TRPC subunits requires specialized experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Use TRPC1 antibodies for immunoprecipitation followed by western blotting with antibodies against other TRPC subunits

  • TRPC1 Antibody (E-6) has been validated for immunoprecipitation applications

  • This approach identifies physical interactions but doesn't necessarily reflect functional channel assemblies

• Electrophysiological characterization:

  • Patch-clamp recording of channel currents with distinctive biophysical properties

  • TRPC1 homomeric channels generally show different conductance, ion selectivity, and voltage dependence compared to heteromeric channels

  • Pharmacological tools (selective channel blockers) can help differentiate channel compositions

• FRET/BRET analysis:

  • Fluorescence or bioluminescence resonance energy transfer between tagged TRPC subunits

  • Requires expression of fluorescently labeled TRPC1 and potential partner subunits

  • Provides evidence of close proximity (≤10 nm) indicative of channel assembly

• Dominant-negative mutant approach:

  • Expression of non-functional TRPC1 mutants can disrupt the function of heteromeric channels containing TRPC1

  • Compare calcium entry in systems expressing wild-type vs. dominant-negative TRPC1

• Differential antibody accessibility:

  • Epitope accessibility may differ between homomeric and heteromeric channels

  • Compare immunostaining patterns using antibodies against different TRPC subunits

These complementary approaches provide a comprehensive understanding of the composition and function of TRPC1-containing channels in specific cellular contexts.

How can researchers address common challenges in TRPC1 antibody-based experiments?

Researchers working with TRPC1 antibodies often encounter specific challenges that require troubleshooting:

• Non-specific binding in Western blots:

  • Increase blocking stringency (5% BSA or milk, with 0.1-0.3% Tween-20)

  • Optimize primary antibody dilution (starting with manufacturer recommendations)

  • Consider using monoclonal antibodies like E-6 for higher specificity

  • Include peptide competition controls to identify non-specific bands

• Weak or absent signal in immunostaining:

  • Optimize antigen retrieval methods for fixed tissues

  • Test different fixation protocols (some epitopes may be fixation-sensitive)

  • Increase antibody concentration or incubation time

  • Try signal amplification systems (tyramide signal amplification, polymeric detection)

  • Confirm TRPC1 expression in the tissue/cells being examined

• Inconsistent results between different antibodies:

  • Different antibodies target distinct epitopes that may have variable accessibility

  • Some epitopes may be masked by protein interactions or post-translational modifications

  • Use KO-validated antibodies when available

  • Compare results with antibodies targeting different regions of TRPC1

• Difficulties in immunoprecipitation:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Cross-linking approaches may help stabilize transient interactions

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Consider using antibody-conjugated beads for cleaner results

• Variability in flow cytometry:

  • Optimize fixation and permeabilization for intracellular epitopes

  • Use directly conjugated antibodies (e.g., RBITC-conjugated) to reduce background

  • Include proper compensation controls when using multiple fluorophores

  • Validate surface expression with membrane-impermeable biotinylation

Addressing these common challenges methodically improves experimental reliability and reproducibility.

What advanced techniques can be used to study TRPC1 dynamics and interactions?

Beyond standard antibody applications, several advanced techniques can provide deeper insights into TRPC1 biology:

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED can resolve TRPC1 localization beyond the diffraction limit

  • Allows visualization of channel clusters and nanoscale organization

  • Requires specialized fluorophore-conjugated antibodies optimized for super-resolution

• Live-cell imaging of TRPC1 dynamics:

  • Antibody fragments (Fab) can be used to track TRPC1 in living cells

  • Alternatively, expression of fluorescently-tagged TRPC1 can be validated using antibodies

  • Enables real-time monitoring of channel trafficking and clustering

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions with spatial resolution (<40 nm)

  • Can confirm TRPC1 interactions with STIM1, Orai1, or other TRPC subunits

  • Uses pairs of antibodies against the two proteins of interest

• Mass spectrometry following immunoprecipitation:

  • Identifies novel TRPC1 interaction partners

  • Can detect post-translational modifications on TRPC1

  • Requires high-quality antibodies suitable for immunoprecipitation, such as the E-6 monoclonal

• Antibody-based calcium imaging:

  • Genetically-encoded calcium indicators fused to anti-TRPC1 antibody fragments

  • Allows measurement of local calcium signals near TRPC1 channels

  • Provides spatial information about calcium signaling microdomains

• Cryo-electron microscopy with antibody labeling:

  • Facilitates structural studies of TRPC1-containing complexes

  • Antibody fragments can help identify TRPC1 within larger assemblies

  • Contributes to understanding channel architecture and gating mechanisms

These advanced approaches extend beyond conventional antibody applications to address sophisticated questions about TRPC1 biology and function.

How can researchers quantitatively assess TRPC1 expression levels across different experimental conditions?

Quantitative assessment of TRPC1 expression requires rigorous methodological approaches:

• Quantitative Western blotting:

  • Include recombinant TRPC1 standard curves for absolute quantification

  • Use housekeeping proteins (β-actin, GAPDH) or total protein staining (Ponceau S) for normalization

  • Employ fluorescent secondary antibodies for wider linear detection range

  • Analyze using specialized software for densitometry

  • Account for the ~91.2 kDa molecular weight of TRPC1

• qRT-PCR validation:

  • Complement protein detection with mRNA quantification

  • Design primers specific to TRPC1 (avoiding other TRPC family members)

  • Validate antibody results by correlating with transcript levels

• Quantitative immunofluorescence:

  • Use standardized image acquisition parameters

  • Include calibration standards in each experiment

  • Employ automated image analysis algorithms to reduce bias

  • Account for tissue autofluorescence and non-specific binding

• Flow cytometry quantification:

  • Use antibodies conjugated to fluorophores like FITC or PE

  • Include calibration beads to convert fluorescence to molecules of equivalent soluble fluorochrome

  • Employ quantitative flow cytometry to determine receptors per cell

• ELISA-based quantification:

  • Develop sandwich ELISA using TRPC1 antibodies recognizing different epitopes

  • Create standard curves with recombinant TRPC1 protein

  • Particularly useful for serum or other fluid samples

These quantitative approaches enable precise comparison of TRPC1 expression across different experimental conditions, tissues, or disease states, facilitating more rigorous statistical analysis and reproducible results.