CHRNA9 Antibody

What is CHRNA9 and why is it significant in research?

CHRNA9 (Cholinergic Receptor, Nicotinic, alpha 9 Neuronal) is a protein that functions as a subunit of nicotinic acetylcholine receptors. This receptor subtype has significant research importance due to its role in auditory processes, pain signaling, and potential involvement in various pathological conditions. The protein has a molecular weight of approximately 54.8 kilodaltons and is encoded by the CHRNA9 gene in humans, which may also be referred to as HSA243342, NACHRA9, or neuronal acetylcholine receptor subunit alpha-9 . Research on CHRNA9 has expanded to include investigations in multiple species, with orthologs identified in canine, porcine, monkey, mouse, and rat models .

What applications are CHRNA9 antibodies commonly used for?

CHRNA9 antibodies are employed in multiple laboratory techniques, with the most common applications being:

• Western blotting (WB) for protein detection and quantification

• Immunohistochemistry (IHC) for tissue localization

• Flow cytometry (FACS) for cell-specific expression analysis

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection

Based on available product information, most commercially available CHRNA9 antibodies have been validated for Western blotting and immunohistochemistry applications . Different antibody products may vary in their application versatility, with some being specifically optimized for particular techniques.

What species reactivity should be considered when selecting a CHRNA9 antibody?

When selecting a CHRNA9 antibody, researchers must carefully consider species reactivity to ensure compatibility with their experimental model. Available CHRNA9 antibodies demonstrate varied cross-reactivity profiles:

When selecting an antibody, researchers should verify that the antibody has been validated in their species of interest, particularly if working with less common research models .

How should CHRNA9 antibodies be stored and handled to maintain activity?

Proper storage and handling of CHRNA9 antibodies are crucial for maintaining their activity and specificity. For lyophilized products such as blocking peptides, the material can typically be stored intact at room temperature for up to two weeks . For longer periods, storage at -20°C is recommended . After reconstitution with appropriate buffers (such as double-distilled water), continued storage at -20°C is generally advised . Researchers should avoid repeated freeze-thaw cycles and follow manufacturer-specific instructions, as storage conditions may vary between different antibody preparations and formulations.

How can the specificity of CHRNA9 antibodies be validated in experimental systems?

Validating the specificity of CHRNA9 antibodies is critical to ensure experimental reliability. Several complementary approaches are recommended:

• Pre-adsorption (blocking peptide) controls: Incubating the antibody with its immunizing peptide before application. If the antibody is specific, this should eliminate or substantially reduce signal in Western blot or immunohistochemistry applications . For example, the Nicotinic Acetylcholine Receptor α9/CHRNA9 Blocking Peptide (BLP-NC019) can be used to validate the Anti-Nicotinic Acetylcholine Receptor α9 (CHRNA9) Antibody (ANC-019) at a ratio of 1 μg peptide per 1 μg antibody .

• Knockout models: Comparing antibody reactivity in wild-type versus CHRNA9 knockout tissues. The constitutive CHRNA9 knockout models have been developed by breeding floxed nAChR α9 heterozygous mice with Cre-expressing mice . These models provide the gold standard for antibody validation.

• Multiple antibody approach: Using different antibodies targeting distinct epitopes of CHRNA9 to verify consistent localization patterns.

• Molecular weight verification: Confirming that the detected protein band matches the expected molecular weight of CHRNA9 (approximately 54.8 kDa) .

What controls should be included in experiments using CHRNA9 antibodies?

Robust experimental design with CHRNA9 antibodies requires several controls:

• Negative controls:

  • Omission of primary antibody while maintaining all other steps

  • Substitution with non-immune IgG from the same species

  • Pre-adsorption controls using specific blocking peptides (e.g., BLP-NC019)

  • Tissue from CHRNA9 knockout animals when available

• Positive controls:

  • Tissues with known CHRNA9 expression (e.g., dorsal root ganglion neurons, as shown in immunohistochemistry results with Anti-Nicotinic Acetylcholine Receptor α9 antibody)

  • Recombinant CHRNA9 protein expression systems

• Loading controls (for Western blot):

  • Housekeeping proteins to normalize for protein loading variations

  • Molecular weight markers to confirm correct band identification

Including these controls helps validate results and troubleshoot potential experimental issues.

What are the optimal dilutions for CHRNA9 antibodies in different applications?

Optimal antibody dilutions vary by application and specific antibody preparation. Based on the available information:

Researchers should optimize dilutions for their specific experimental conditions through titration experiments. The optimal dilution balances signal strength against background and non-specific binding.

How do different CHRNA9 antibodies compare in epitope recognition and functional applications?

CHRNA9 antibodies target different epitopes of the protein, which can significantly impact their utility in specific applications:

The epitope location can affect antibody performance in applications where protein conformation matters (e.g., immunoprecipitation, flow cytometry). N-terminal antibodies may be advantageous for detecting full-length proteins, while antibodies targeting conserved regions may provide better cross-species reactivity.

How can researchers distinguish between CHRNA9 and other nicotinic acetylcholine receptor subunits?

Distinguishing between closely related nicotinic acetylcholine receptor subunits requires careful antibody selection and experimental design:

• Epitope selectivity: Choose antibodies targeting unique regions of CHRNA9 not conserved in other subunits. The N-terminal region (AA 8-42) targeted by some antibodies like ABIN1944733 may provide subunit specificity .

• Validation in knockout models: CHRNA9 knockout models provide the most definitive validation of antibody specificity . When screening tissue from these models, truly specific antibodies should show no signal in the knockout samples.

• Co-localization studies: Combining CHRNA9 antibodies with antibodies against other subunits (especially α10, which often partners with α9) can help determine receptor composition.

• Cross-reactivity testing: Systematic testing against recombinant proteins of different nicotinic receptor subunits can identify potential cross-reactivity.

• Mass spectrometry verification: Following immunoprecipitation, mass spectrometry analysis can confirm the identity of the precipitated protein.

What methodological approaches can resolve contradictory findings using different CHRNA9 antibodies?

When different CHRNA9 antibodies yield contradictory results, several methodological approaches can help resolve discrepancies:

• Systematic epitope mapping: Determine exactly which regions of CHRNA9 each antibody recognizes, and consider how protein conformation or post-translational modifications might affect epitope accessibility.

• Multi-technique validation: Employ complementary techniques (e.g., immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization) to cross-validate findings.

• Genetic approaches: Use RNA interference, CRISPR-Cas9 gene editing, or genetic knockout models to manipulate CHRNA9 expression and correlate with antibody signal changes .

• Blocking peptide experiments: Test specificity using pre-adsorption with the immunizing peptide for each antibody . True signals should be abolished, while non-specific binding may persist.

• Functional correlation: Correlate antibody labeling with functional assays of CHRNA9 activity (e.g., electrophysiology in expression systems).

• Advanced imaging: Super-resolution microscopy combined with proximity ligation assays can provide additional evidence for true co-localization versus coincidental overlap.

What are common causes of false positive or false negative results with CHRNA9 antibodies?

Understanding potential sources of error helps researchers interpret results accurately:

Causes of false positive results:

• Cross-reactivity with other nicotinic receptor subunits due to sequence homology

• Non-specific binding to unrelated proteins with similar epitopes

• Excessively high antibody concentrations leading to off-target binding

• Inadequate blocking or inappropriate blocking reagents

• Endogenous peroxidase or phosphatase activity in immunohistochemistry

• Autofluorescence in certain tissues (especially fixed tissues)

Causes of false negative results:

• Epitope masking due to fixation, particularly with formalin fixation

• Protein denaturation affecting conformation-dependent epitopes

• Insufficient antigen retrieval in fixed tissues

• Degradation of the target protein during sample preparation

• Suboptimal antibody concentration or incubation conditions

• Species mismatch between antibody specificity and experimental samples

How can researchers optimize Western blot protocols for CHRNA9 detection?

Optimizing Western blot protocols for CHRNA9 detection involves several technical considerations:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Consider membrane-enriched fractions, as CHRNA9 is a membrane protein

  • Avoid excessive heating, which may cause aggregation of membrane proteins

• Protein separation:

  • Use 8-10% SDS-PAGE gels for optimal resolution around 54.8 kDa

  • Include positive controls from tissues known to express CHRNA9

• Transfer conditions:

  • Employ wet transfer for membrane proteins

  • Consider using PVDF membranes, which may provide better protein retention

• Blocking and antibody incubation:

  • Test multiple blocking solutions (BSA vs. milk proteins)

  • Optimize primary antibody dilution (typically starting at 1:400)

  • Consider longer incubation times at 4°C to improve specific binding

• Detection system:

  • Enhanced chemiluminescence (ECL) systems provide good sensitivity

  • Consider more sensitive detection for low-abundance expression

What strategies improve immunohistochemical detection of CHRNA9 in different tissue types?

Successful immunohistochemical detection of CHRNA9 requires tissue-specific optimization:

• Fixation considerations:

  • Compare paraformaldehyde fixation versus frozen sections

  • For paraformaldehyde-fixed tissues, optimize fixation time to balance antigen preservation and tissue morphology

• Antigen retrieval methods:

  • Test heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Compare with enzymatic retrieval methods if heat-based approaches fail

  • Optimize retrieval times for specific tissues

• Signal enhancement:

  • Consider tyramide signal amplification for low-abundance targets

  • Use neuron-specific counterstains (e.g., NeuN) to confirm neuronal localization

  • When expecting sparse expression, use DAPI counterstain to provide context as demonstrated in rat DRG sections

• Background reduction:

  • Include endogenous peroxidase quenching step for HRP-based detection

  • Add avidin/biotin blocking for biotin-based detection systems

  • Use specific blocking peptides as pre-adsorption controls

• Tissue-specific considerations:

  • For neuronal tissues (such as DRG), ensure proper tissue preservation to maintain cellular morphology

  • For inner ear tissues, where CHRNA9 is physiologically relevant, specialized fixation protocols may be necessary

How can CHRNA9 antibodies be integrated into multi-protein analysis workflows?

Integrating CHRNA9 antibodies into multi-protein analysis provides more comprehensive insights:

• Multiplex immunofluorescence:

  • Combine CHRNA9 antibodies with markers for specific cell types

  • Use antibodies raised in different host species to allow simultaneous detection

  • Consider spectral unmixing approaches for tissues with autofluorescence

• Co-immunoprecipitation studies:

  • Use CHRNA9 antibodies to pull down receptor complexes

  • Analyze interacting proteins through proteomics approaches

  • Confirm specific interactions through reverse co-immunoprecipitation

• Proximity ligation assays:

  • Investigate protein-protein interactions between CHRNA9 and potential binding partners

  • Particularly useful for studying α9/α10 receptor assembly

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies allow high-dimensional analysis of protein expression

  • Enables correlation of CHRNA9 expression with numerous other proteins simultaneously

• Single-cell analysis pipelines:

  • Combine antibody-based protein detection with single-cell transcriptomics

  • Correlate protein expression with transcriptional profiles

What are recommended experimental designs for studying CHRNA9 expression in disease models?

When investigating CHRNA9 in disease contexts, robust experimental design is essential:

• Control selection:

  • Include age-matched, sex-matched controls

  • For cancer studies, compare paired tumor/normal tissue from the same patient

  • Consider both positive controls (tissues known to express CHRNA9) and negative controls (CHRNA9 knockout tissues if available)

• Quantification approaches:

  • Use digital image analysis for immunohistochemistry quantification

  • Consider multiplex immunofluorescence to correlate with disease markers

  • Employ RT-qPCR to correlate protein expression with mRNA levels

• Temporal considerations:

  • Analyze multiple time points in progressive disease models

  • For interventional studies, include pre-treatment baselines

• Validation across models:

  • Compare findings across multiple model systems (cell lines, animal models, human samples)

  • Use conditional knockout models to assess tissue-specific effects

• Functional correlation:

  • Link expression changes to functional outcomes

  • Consider electrophysiological studies to assess receptor function

How can genetic tools complement antibody-based CHRNA9 research?

• CRISPR-Cas9 gene editing:

  • Generate knockout or knock-in cell lines for antibody validation

  • Create epitope-tagged versions of CHRNA9 for antibody-independent detection

• Conditional knockout models:

  • Use tissue-specific or inducible Cre recombinase systems with floxed CHRNA9 alleles

  • Enables temporal and spatial control of gene deletion

• Transgenic reporter systems:

  • Generate CHRNA9-GFP fusion proteins or promoter-reporter constructs

  • Provides complementary detection method to antibody staining

• RNA interference:

  • Use siRNA or shRNA to temporarily reduce CHRNA9 expression

  • Compare antibody signal reduction with knockdown efficiency

• Single-cell transcriptomics:

  • Correlate protein detection with mRNA expression at single-cell resolution

  • Identify cell populations with discordant protein/mRNA expression