CHRNB2 Antibody

What is CHRNB2 and why is it important in neurological research?

CHRNB2 (cholinergic receptor, nicotinic, beta 2) is a crucial neuronal subunit of nicotinic acetylcholine receptors (nAChRs). These receptors are ligand-gated ion channels that allow the flow of sodium and potassium across the plasma membrane in response to ligands such as acetylcholine and nicotine . CHRNB2 plays critical roles in brain function as part of the abundant neuronal nAChRs in the central nervous system . Its importance in neurological research stems from its association with several conditions, including autosomal dominant nocturnal frontal lobe epilepsy . After binding acetylcholine, the AChR undergoes an extensive conformational change affecting all subunits, leading to the opening of an ion-conducting channel across the plasma membrane that is permeable to sodium ions .

What are the primary types of CHRNB2 antibodies available for research?

Research-grade CHRNB2 antibodies are available in several formats with varying specifications:

These antibodies target different epitopes of the CHRNB2 protein, including extracellular domains and cytoplasmic regions, making them suitable for various experimental applications .

How can I validate the specificity of a CHRNB2 antibody?

Validating CHRNB2 antibody specificity requires a multi-step approach:

• Western blot analysis: Compare bands from target tissues (e.g., SH-SY5Y cells, mouse brain tissue) with the expected molecular weight of CHRNB2 (calculated MW: 57 kDa, observed MW: varying between 48-65 kDa depending on the antibody) .

• Peptide blocking experiments: Pre-incubate the antibody with a specific blocking peptide (like the Nicotinic Acetylcholine Receptor β2/CHRNB2 extracellular blocking peptide) and compare results with non-blocked antibody .

• Immunohistochemistry with positive controls: Use tissues known to express CHRNB2, such as human brain tissue, particularly cortex regions .

• Cross-reactivity testing: Examine reactivity across multiple species using BLAST analysis to confirm antibody specificity (most CHRNB2 antibodies show high sequence homology across human, mouse, and rat) .

For most rigorous validation, a combination of these methods should be employed to ensure antibody specificity before proceeding with experimental applications.

What are the optimal conditions for Western blot analysis using CHRNB2 antibodies?

Optimal Western blot conditions for CHRNB2 antibodies typically include:

Sample preparation:

• SH-SY5Y cells, mouse brain tissue, or human cerebellum lysate (in RIPA buffer) are suitable positive controls

• Load approximately 35 μg of protein per lane

Dilution ratios:

• Most CHRNB2 antibodies perform well at dilutions between 1:200-1:2000

• Specific recommendations:

  • Polyclonal antibodies (17844-1-AP): 1:200-1:1000

  • Monoclonal antibodies: 1:500-1:2000

Expected results:

• Predicted molecular weight: 57 kDa

• Observed molecular weight: Varies between antibodies (48-65 kDa)

  • Example: 17844-1-AP detects bands at 60-65 kDa

  • Some antibodies detect bands at 48-52 kDa

Blocking and incubation:

• Standard blocking with 5% non-fat milk or BSA

• Optimal incubation time: room temperature for 1.5 hours or overnight at 4°C

For troubleshooting, it's recommended to titrate the antibody in each testing system to obtain optimal results, as the optimal dilution can be sample-dependent .

How should I perform immunohistochemistry with CHRNB2 antibodies?

For optimal immunohistochemistry (IHC) results with CHRNB2 antibodies:

Tissue preparation:

• Formalin-fixed, paraffin-embedded tissues (especially human brain tissue)

• Fresh-frozen sections may also be suitable for some antibodies

Antigen retrieval:

• Primary recommendation: TE buffer pH 9.0

• Alternative: Citrate buffer pH 6.0

Antibody dilutions:

• Polyclonal antibodies: 1:50-1:500

• For specific antibodies like ab189174: 3.75 μg/ml

Detection systems:

• Standard avidin-biotin complex (ABC) or polymer-based detection systems

• For fluorescent detection, secondary antibodies conjugated to fluorophores like AlexaFluor-594

Positive controls:

• Human brain tissue, particularly cortex regions

• SH-SY5Y cells for immunocytochemistry

Protocols:
Follow manufacturer-specific protocols, such as the IHC protocol for CHRNB2 antibody 17844-1-AP . For flow cytometry applications, a dilution of 1:200-1:400 is typically recommended for optimal staining .

What cell models are most appropriate for studying CHRNB2 expression and function?

Several cell models have been validated for CHRNB2 research:

Neuronal cell lines:

• SH-SY5Y human neuroblastoma cells: Widely used and documented positive control for CHRNB2 expression

• PC12 rat pheochromocytoma cells: Suitable for cell surface detection of nAChR β2 in live cell imaging experiments

Heterologous expression systems:

• HEK293 cells transfected with CHRNB2-hIgGFc: Useful for antibody validation and functional studies

• CBA (cell-based assay) with α4β2-nAChR–transfected cells: For detecting antibodies against extracellular domains of nAChR subunits

Primary cultures:

• Mouse brain tissue: Particularly useful for knockout studies comparing wild-type and Chrnb2−/− mutants

For optimal expression of nAChRs in heterologous systems, co-transfection with chaperone proteins NACHO and RIC3 can significantly improve surface expression levels. Treatment with 1mM nicotine or 5mM DFMO ligands further enhances expression, as demonstrated in recent research on developing sensitive cell-based assays .

How can CHRNB2 antibodies be used to investigate autoimmune encephalitis syndromes?

CHRNB2 antibodies play a critical role in investigating autoimmune encephalitis syndromes (AES):

Development of diagnostic assays:

• Cell-based assays (CBAs) using α4β2-nAChR-transfected cells can selectively detect potentially pathogenic antibodies targeting major neuronal nAChR subtypes

• Flow cytometry (FACS) can confirm CBA findings, while indirect immunohistochemistry (IHC) can investigate autoantibody binding to brain tissue

Enhanced detection methodology:
Recent research has focused on improving surface expression of α4β2-nAChRs to develop more sensitive and accurate live-CBAs by:

• Co-transfecting nAChRs with NACHO and RIC3 chaperons

• Treating cell cultures with 1mM nicotine or 5mM DFMO ligands

Clinical applications:
In a study of 1752 patients requiring testing for AES-associated antibodies, three patients were identified with antibodies specifically targeting the α4 subunit of α4β2-nAChR, demonstrating the utility of these techniques in identifying previously undiagnosed cases .

This methodology is particularly valuable for investigating "orphan" AES cases where patients have yet to be identified for specific autoantibodies, potentially leading to improved diagnosis and treatment options.

What approaches can be used to study gene-gene interactions involving CHRNB2?

Research into gene-gene interactions involving CHRNB2 can employ several sophisticated approaches:

SNP selection and genotyping:

• Select SNPs based on high heterozygosity (minor allele frequency ≥ 0.05) and uniform coverage of CHRNB2

• For comprehensive studies, include multiple SNPs (e.g., 4 SNPs for CHRNB2 as used in gene interaction studies)

Statistical interaction analysis:
Research has demonstrated significant statistical interactions between CHRNB2 and other genes related to neuronal function:

• CHRNB2 and CHRNA4: Prediction accuracy 0.565-0.593, empirical P < 0.01

• CHRNB2 and NTRK2: Prediction accuracy 0.565-0.593, empirical P < 0.01

• CHRNB2 and BDNF: Prediction accuracy unreported, empirical P = 0.068

Ethnic considerations:
Gene-gene interactions may show varying significance in different ethnic samples, suggesting the importance of analyzing combined samples as well as ethnically-stratified data .

These methodologies provide valuable tools for investigating the complex genetic networks involving CHRNB2, particularly in conditions like nicotine dependence where multiple genes and their interactions influence phenotypic expression.

How can CHRNB2 knockout models advance our understanding of neuronal nicotinic acetylcholine receptor function?

CHRNB2 knockout (Chrnb2−/−) models provide powerful tools for investigating nAChR function:

Transcriptomic analysis:

• Microarray technology can compare gene expression in the retina and lateral geniculate nucleus (LGN) of Chrnb2−/− mutants with wild-type animals

• This approach reveals molecular changes underlying structural and functional abnormalities in visual systems of Chrnb2−/− mutants

Developmental studies:

• Chrnb2−/− models help delineate the role of nAChRs during critical developmental periods

• Research shows that despite normal retinal appearance in Chrnb2−/− mutants, altered gene expression contributes to abnormal projection patterns of retinal ganglion cells to the LGN

Genetic background effects:

• Comparison of transcriptomes between different Chrnb2−/− mutant strains reveals how genetic background influences gene expression

• This provides insights into the complex interplay between neural activity and molecular expression

These models facilitate distinguishing between abnormalities driven by altered retinal activity during development versus those resulting from aberrant molecular expression, advancing our understanding of how nAChRs contribute to neural circuit formation and function.

What factors affect the observed molecular weight of CHRNB2 in Western blot analysis?

Several factors can cause variations in the observed molecular weight of CHRNB2 in Western blot analyses:

Expected versus observed weights:

• Calculated molecular weight: 57 kDa (502 amino acids)

• Observed molecular weights vary significantly:

  • 60-65 kDa (17844-1-AP antibody)

  • 48 kDa (ab189174 antibody)

  • 50-52 kDa (other antibodies)

Contributing factors:

• Post-translational modifications: Glycosylation and phosphorylation can increase the apparent molecular weight

• Tissue/sample source: Different expression systems or tissues may produce variants with different modifications:

  • SH-SY5Y cells

  • Mouse brain tissue

  • Human cerebellum lysate

• SDS-PAGE conditions: Gel percentage, running buffer composition, and voltage can affect protein migration

• Antibody specificity: Different antibodies recognize different epitopes which may be differentially accessible based on protein folding or modification

For accurate interpretation, researchers should always include appropriate positive controls (such as SH-SY5Y cells) and compare results with the specific antibody's validation data from the manufacturer .

How can I optimize immunocytochemistry protocols for detecting CHRNB2 in live cells?

Optimizing immunocytochemistry protocols for live-cell detection of CHRNB2 requires special considerations:

Cell preparation and fixation:

• For live cell detection: Use intact cells without fixation (e.g., PC12 cells)

• For fixed cell protocols: 100% methanol fixation (5 min) has been validated with SH-SY5Y cells

Blocking and permeabilization:

• For fixed cells: 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween (1 hour)

• For live cells: Avoid permeabilization reagents to maintain membrane integrity

Antibody selection and dilution:

• Choose antibodies targeting extracellular epitopes for live cell staining

• Anti-Nicotinic Acetylcholine Receptor β2 (extracellular) Antibody (#ANC-012): 1:100 dilution

• For SH-SY5Y cells with fixed protocols: 1:1000 dilution (ab41174)

Detection systems:

• For fluorescent detection: Secondary antibodies like goat anti-rabbit-AlexaFluor-594 (red) for extracellular domain antibodies

• Optional counterstaining: Cell nuclei visualization using Hoechst 33342 (blue)

• Membrane visualization: Alexa Fluor® 594 WGA (1:200 dilution, 1 hour)

Visualization:

• For live cells: Maintain physiological conditions during imaging

• For fixed cells: Standard fluorescence microscopy protocols apply

These optimized protocols enable accurate detection of CHRNB2 in both live and fixed cellular contexts, supporting a range of experimental applications from receptor trafficking to co-localization studies.

What strategies can address cross-reactivity or non-specific binding with CHRNB2 antibodies?

To address cross-reactivity or non-specific binding issues with CHRNB2 antibodies:

Validation with peptide blocking:

• Pre-incubate antibody with Nicotinic Acetylcholine Receptor β2/CHRNB2 (extracellular) Blocking Peptide (#BLP-NC012)

• Compare staining patterns between blocked and non-blocked antibody samples

Species cross-reactivity assessment:

• Consult BLAST analysis data for sequence homology across species

• High sequence homology exists between human, chimpanzee, gorilla (100%), and mouse, rat, dog, bovine, pig (92%)

• Choose antibodies with validated reactivity for your species of interest

Optimize blocking conditions:

• For Western blot: Test alternative blocking agents (milk vs. BSA)

• For immunohistochemistry/immunocytochemistry: Increase blocking time or concentration

Antibody dilution optimization:

• Perform titration experiments to determine optimal antibody concentration

• Follow manufacturer recommendations as starting points:

  • Western blot: 1:200-1:2000

  • IHC: 1:50-1:500

  • IF/ICC: 1:50-1:200

Alternative antibody selection:

• When persistent cross-reactivity occurs, consider alternative antibodies targeting different epitopes

• Compare monoclonal (higher specificity) vs. polyclonal (higher sensitivity) options based on experimental needs

Using these strategies can significantly improve specificity and reduce background in CHRNB2 antibody applications, leading to more reliable and reproducible research outcomes.

How might CHRNB2 antibodies contribute to understanding neurodegenerative disorders?

CHRNB2 antibodies offer significant potential for advancing neurodegenerative disorder research:

Receptor expression profiling:

• Quantitative analysis of nAChR β2 subunit expression changes in disease states

• Comparing expression patterns between healthy and pathological tissues using Western blot and immunohistochemistry

Autoimmunity investigation:

• Detection of endogenous autoantibodies targeting nAChRs in neurological disorders

• Expanding upon recent research identifying antibodies to α4β2-nAChR in previously seronegative cases of autoimmune CNS disorders

Therapeutic target validation:

• Evaluating the efficacy of drugs targeting α4β2-nAChRs in neurodegenerative disorders

• Monitoring changes in receptor expression and distribution following therapeutic intervention

Disease mechanism elucidation:

• Investigating potential connections between nAChR dysfunction and neurodegenerative processes

• Building on established associations, such as between CHRNB2 mutations and autosomal dominant nocturnal frontal lobe epilepsy

These applications could significantly enhance our understanding of conditions like Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders where cholinergic system dysfunction is implicated.

What novel detection methods might improve sensitivity and specificity for CHRNB2 research?

Emerging technologies show promise for enhancing CHRNB2 detection:

Enhanced cell-based assays:

• Co-expression with chaperone proteins (NACHO and RIC3) significantly improves surface expression of nAChRs

• Treatment with specific ligands (1mM nicotine or 5mM DFMO) further enhances expression for more sensitive detection

Multiplexed detection systems:

• Simultaneous detection of multiple nAChR subunits (α4, β2, α7) to study receptor assembly and stoichiometry

• Combined with super-resolution microscopy for nanoscale visualization of receptor distribution

Single-molecule imaging techniques:

• Direct stochastic optical reconstruction microscopy (dSTORM) or photoactivated localization microscopy (PALM) for studying CHRNB2 dynamics at the single-molecule level

• Quantum dot-conjugated antibodies for long-term tracking of receptor movement in live cells

Proximity-based assays:

• Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) to study protein-protein interactions involving CHRNB2

• Proximity ligation assays (PLA) for visualizing protein complexes containing CHRNB2 in situ

These advanced methodologies could overcome current limitations in sensitivity and specificity, enabling more detailed characterization of CHRNB2 expression, localization, and function in complex neuronal systems.

How can CHRNB2 antibodies be used to investigate receptor trafficking and membrane dynamics?

CHRNB2 antibodies enable sophisticated studies of receptor trafficking and membrane dynamics:

Live cell surface labeling:

• Antibodies targeting extracellular domains (like ANC-012) can be used to label surface-expressed receptors in live cells

• This approach has been successfully employed with PC12 cells to visualize cell surface expression of nAChR β2

Receptor internalization studies:

• Sequential labeling with different fluorophore-conjugated antibodies before and after stimulation

• Measuring changes in surface-to-intracellular receptor ratios following agonist exposure

Receptor assembly and transport:

• Immunoprecipitation with CHRNB2 antibodies followed by Western blot analysis for associated proteins

• Building on research showing that Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

Subcellular localization:

• Immunofluorescence co-localization studies with organelle markers

• Examining differences in receptor distribution between neuronal compartments (soma vs. dendrites vs. axons)

These techniques provide critical insights into the cellular mechanisms controlling nAChR availability and function, which may be disrupted in neurological disorders.

What considerations are important when developing assays to detect autoantibodies against CHRNB2?

Developing assays for autoantibody detection against CHRNB2 requires several important considerations:

Cell-based assay optimization:

• Use live cell-based assays (CBAs) rather than fixed cells to preserve native epitope conformation

• Enhance surface expression of nAChRs through co-transfection with RIC3 and NACHO chaperones

• Further increase expression by including nicotine or DFMO in cell culture medium

Validation approaches:

• Confirm CBA findings using flow cytometry (FACS)

• Validate with indirect immunohistochemistry (IHC) on rat brain tissue to examine binding patterns

Epitope targeting:

• Focus detection on extracellular domains of CHRNB2, as these are accessible to circulating antibodies

• Design assays that can distinguish between antibodies targeting different subunits (e.g., α4 vs. β2)

Control populations:

• Include appropriate control groups (e.g., healthy controls and patients with other neuropsychiatric diseases)

• In one study, researchers screened 1752 patients suspected of autoimmune encephalitis alongside 1203 "control" patients with other neuropsychiatric diseases

Clinical correlation:

• Correlate antibody detection with clinical presentations

• Document cases that are positive for CHRNB2 antibodies but negative for other established autoantibodies

These methodological considerations have proven effective in identifying previously unrecognized autoantibodies in patients with suspected autoimmune encephalitis syndromes.