CHRNA7 Antibody

What is CHRNA7 and why is it an important target for antibody development?

CHRNA7 (cholinergic receptor, nicotinic, alpha 7) is a neuronal nicotinic acetylcholine receptor subunit that forms homo-oligomeric ligand-gated ion channels mediating fast signal transmission at synapses. It is crucial for research because it displays marked permeability to calcium ions and is a major component of brain nicotinic receptors that are blocked by alpha-bungarotoxin . The protein has a calculated molecular weight of 56 kDa (502 amino acids) and is widely expressed in multiple tissues .

CHRNA7 plays important physiological roles in:

• Synaptic transmission in the central nervous system

• Regulation of inflammatory responses through the cholinergic anti-inflammatory pathway

• Calcium signaling within neurons and immune cells

The development of specific antibodies against CHRNA7 is essential for studying its expression, localization, and function in normal physiology and disease states, particularly in neuropsychiatric disorders and inflammatory conditions .

What are the fundamental differences between CHRNA7 and CHRFAM7A that researchers must understand?

This is a critical distinction that impacts experimental design and interpretation:

CHRNA7:

• Codes for the full alpha-7 nicotinic acetylcholine receptor subunit (502 amino acids)

• Forms functional homo-oligomeric ion channels

• Present in humans and other species (mouse, rat, etc.)

• Mediates calcium influx and signal transduction

CHRFAM7A:

• Human-specific chimeric gene formed by partial duplication of CHRNA7 (exons 5-10) fused with FAM7A (exons A-E)

• Acts as a dominant negative modulator of CHRNA7 function

• Produces protein that lacks much of the ligand-binding domain but retains the transmembrane region

• When co-expressed with CHRNA7, reduces ACh-evoked currents without reducing receptor binding

• Creates "ACh-silent" receptors (bind ligands but don't function normally)

This distinction is crucial because:

• CHRFAM7A is human-specific and absent in laboratory animals

• Most antibodies cannot distinguish between these proteins

• Expression ratios of CHRFAM7A/CHRNA7 increase during inflammation

• Findings from animal models may not translate directly to humans due to this regulatory mechanism

How can researchers validate the specificity of CHRNA7 antibodies?

Validating CHRNA7 antibody specificity requires a multi-pronged approach:

Use of genetic controls:
The gold standard is testing on CHRNA7 knockout tissues or cells. A comprehensive study evaluating nine different antibodies found that most still stained positively in knockout mice, indicating lack of specificity. Only one antibody (ab23832) showed clear specificity by not labeling nerve fibers in knockout mice .

Molecular characterization:

• Western blot analysis to confirm detection of proteins at the expected 56 kDa

• 2D electrophoresis for higher resolution protein separation

• Mass spectrometry on immunoprecipitated samples to confirm identity

RNA expression correlation:

• Compare antibody staining patterns with mRNA expression by qRT-PCR

• Use specific primer sets:

  • For CHRNA7: primers spanning exons 3-4 (e.g., Hs01063372_m1)

  • For CHRFAM7A: primers spanning exons 1-2 (e.g., Hs00415199_m1)

Cross-reactivity assessment:
2D electrophoresis and mass spectrometry have identified cross-reactivity of some CHRNA7 antibodies with β-actin and β-enolase, which can be confirmed by double immunolabeling .

Implementing these validation steps is essential before conducting experiments using CHRNA7 antibodies, as many commercially available antibodies lack sufficient specificity when rigorously tested.

What epitope considerations are critical when selecting CHRNA7 antibodies for human versus animal studies?

When selecting CHRNA7 antibodies, epitope targeting is a crucial consideration that directly impacts experimental interpretation:

For human studies:

• Antibodies targeting amino acids 1-100 (exons 1-4) may detect both CHRNA7 and CHRFAM7A

• Those targeting the deleted region in CHRFAM7A (e.g., exon 10, amino acids 367-502) are more specific for CHRNA7

• Consider whether distinguishing between CHRNA7 and CHRFAM7A is important for your research question

• Examples from literature:

  • Antibody ab23832 targets amino acids 1-100 (not in deleted region)

  • Antibodies ab28741, sc-5544, and AS-5631S target regions within exon 10 (deleted in CHRFAM7A)

For animal studies:

Experimental validation table from published research:

When possible, researchers should validate antibodies in their specific experimental system using appropriate controls .

How does the human-specific CHRFAM7A gene impact translational research from animal models to human applications?

The human-specific CHRFAM7A gene creates significant translational challenges that researchers must consider:

Unique receptor regulation:
CHRFAM7A acts as a dominant negative modulator of CHRNA7 function, creating "ACh-silent" receptors that bind ligands but don't function normally. When co-expressed with CHRNA7, it causes significant reduction of ACh-evoked currents without reducing receptor binding .

Inflammatory response differences:

• The CHRFAM7A/CHRNA7 ratio increases in human macrophages stimulated with LPS

• This ratio is elevated in subjects with chronic inflammatory diseases

• May be increased in patients with schizophrenia and bipolar disorder

Drug development implications:

• Compounds targeting CHRNA7 may show different efficacy in humans vs. animal models

• Allosteric modulators like PNU-120596 show larger increases in ACh-evoked current in cells expressing CHRFAM7A

• Animal models lacking CHRFAM7A may not accurately predict human responses to CHRNA7-targeted therapeutics

Experimental approaches to address this challenge:

• Humanized mouse models expressing CHRFAM7A

• In vitro studies co-expressing CHRFAM7A with CHRNA7

• Specific detection methods to distinguish between CHRNA7 and CHRFAM7A expression

• Measuring CHRFAM7A/CHRNA7 ratios when assessing inflammatory states in human samples

This human-specific regulation mechanism highlights why caution is needed when extrapolating findings from animal models to human applications, particularly for therapeutic development targeting this receptor system.

What is the optimal antigen retrieval protocol for CHRNA7 immunohistochemistry?

Antigen retrieval is critical for successful CHRNA7 immunohistochemistry. Based on published protocols, the following methods are recommended:

Primary recommendation:

• Buffer: TE buffer pH 9.0 (alkaline retrieval)

• Heat method: Microwave or pressure cooker

• Duration: 10-20 minutes (microwave) or 3-5 minutes (pressure cooker)

Alternative method:

• Buffer: Citrate buffer pH 6.0 (acidic retrieval)

• Similar heating parameters as above

Tissue-specific considerations:

• Brain tissue (high CHRNA7 expression) may require gentler retrieval to preserve morphology

• Human brain tissue shows positive IHC detection with both primary and alternative protocols

• Mouse brain tissue similarly shows good results with these methods

Post-retrieval processing:

• Allow slides to cool slowly to room temperature (15-20 minutes)

• Wash thoroughly in buffered solution before proceeding with blocking

• Block endogenous peroxidase activity when using HRP detection systems

Validation approach:
Always perform parallel experiments with positive control tissues (e.g., brain tissue) and negative controls (primary antibody omission and ideally knockout tissue when available) .

The optimization of antigen retrieval methods significantly impacts staining intensity and specificity, and should be carefully titrated for each experimental system.

How should Western blot protocols be optimized for CHRNA7 detection?

Western blot detection of CHRNA7 requires specific technical considerations:

Sample preparation:

• Use specialized lysis buffers that effectively solubilize membrane proteins (CHRNA7 is a transmembrane protein)

• Include protease inhibitors to prevent degradation

• Keep samples cold during processing

• Validated positive control samples include: Jurkat cells, rat brain tissue, mouse skeletal muscle tissue, MCF-7 cells, and SH-SY5Y cells

Electrophoresis and transfer:

• Expected molecular weight: 56 kDa

• Recommended protein amount: 20-50 µg per lane

• For 2D electrophoresis: First dimension pH range 3-10, second dimension 12% SDS-PAGE

• Transfer to PVDF membranes using tank blotting system

• For human samples, be aware that CHRNA7 and CHRFAM7A may appear at similar molecular weights

Blocking and antibody incubation:

• Blocking: Roti-Block or 5% non-fat milk in PBS-T (PBS containing 0.05% Tween 20)

• Primary antibody dilutions: 1:500-1:2000 for most CHRNA7 antibodies

• Incubation: Overnight at 4°C for primary antibody

• Washing: 5 times for 3 minutes with PBS-T

• Secondary antibody: HRP-conjugated, 1:3000 dilution, 1 hour at room temperature

Detection and troubleshooting:

• Enhanced chemiluminescence (ECL SuperSignal kit) works well for detection

• If multiple bands appear, consider cross-reactivity with other nAChR subunits or CHRFAM7A

• Validation with knockout samples is ideal but not always available

• Cross-reactivity with β-actin (42 kDa) and β-enolase has been documented

Published protocol reference:
For detailed 2D electrophoresis protocol that identified cross-reactivity issues, refer to the methods of Moser et al. (2015), which effectively separated and identified CHRNA7 cross-reactive proteins .

What specific techniques can distinguish between CHRNA7 and CHRFAM7A expression in human samples?

Distinguishing between CHRNA7 and CHRFAM7A expression in human samples requires specialized approaches:

RNA detection methods:

• Quantitative real-time PCR with specific primers:

  • For CHRNA7: primers spanning exons 3-4 (Hs01063372_m1)

  • For CHRFAM7A: primers spanning exons 1-2 of the hybrid gene (Hs00415199_m1)

  • Calculate the CHRFAM7A/CHRNA7 ratio as a meaningful measure of inflammatory state

Protein detection strategies:

• Western blot with epitope-specific antibodies:

  • Use antibodies targeting regions present in CHRNA7 but absent in CHRFAM7A (e.g., amino acids 367-502)

  • Consider protein size differences, though both may appear around 56 kDa

  • Perform parallel detection with antibodies targeting shared and unique regions

• 2D gel electrophoresis:

  • Offers better resolution of proteins with similar molecular weights

  • Follow with mass spectrometry to confirm protein identity

Functional assays:

• Electrophysiology:

  • CHRNA7 forms functional ion channels while CHRFAM7A does not

  • Co-expression reduces ACh-evoked currents

  • Patch-clamp recordings can distinguish functional from non-functional receptors

• Calcium imaging:

  • CHRNA7 activation increases intracellular calcium

  • CHRFAM7A expression modulates this response

Pharmacological tools:

• α-Bungarotoxin binding:

  • Radiolabeled α-bungarotoxin (I-BTX) binds functional CHRNA7

  • Co-expression with CHRFAM7A shows binding but reduced function

• Allosteric modulators:

  • PNU-120596 shows larger current increases in cells expressing both proteins compared to CHRNA7 alone

These specialized techniques are essential for accurate characterization of the human-specific regulatory mechanism involving these two related proteins.

What are the most common causes of false positive and false negative results with CHRNA7 antibodies?

Understanding the causes of unreliable results is essential for experimental design and interpretation:

Common causes of false positives:

Common causes of false negatives:

Validation strategies to minimize both issues:

• Use gene-deficient (knockout) tissues as gold standard negative controls

• Include positive control tissues with confirmed expression

• Compare multiple antibodies targeting different epitopes

• Correlate protein detection with mRNA expression analysis

• Employ functional assays to confirm biological activity

The study by Moser et al. (2015) demonstrated that nine different commercial antibodies for CHRNA7 and CHRM3 showed varying specificity when rigorously tested against knockout tissues, highlighting the importance of thorough validation .

How can researchers optimize CHRNA7 antibody dilutions for different applications and tissue types?

Optimization of CHRNA7 antibody dilutions requires systematic titration and consideration of specific factors for each application:

Western Blot (WB):

Immunohistochemistry (IHC):

Methodology for antibody titration:

• Prepare a dilution series (e.g., 1:50, 1:100, 1:200, 1:500)

• Process all samples identically except for antibody concentration

• Include positive and negative controls with each dilution

• Evaluate:

  • Signal-to-noise ratio

  • Specificity of staining pattern

  • Reproducibility across replicates

• Select the highest dilution that maintains specific signal while minimizing background

Additional considerations:

• Sample-dependent optimization is essential; "check data in validation data gallery" for sample-specific recommendations

• Different antibody clones may require different dilutions (monoclonal vs. polyclonal)

• Detection system sensitivity affects optimal dilution (HRP vs. fluorescent)

• Protein expression levels vary across tissues and experimental conditions

Manufacturers recommend that "this reagent should be titrated in each testing system to obtain optimal results" , highlighting the importance of optimization for each specific experimental setup.

What are the critical control experiments needed when working with CHRNA7 antibodies?

Robust control experiments are essential for reliable interpretation of CHRNA7 antibody results:

Essential negative controls:

Essential positive controls:

Application-specific controls:

For Western Blot:

• Loading control (β-actin, GAPDH) - caution with β-actin as some CHRNA7 antibodies cross-react

• Molecular weight marker to confirm 56 kDa band

• Positive control lysate (e.g., brain tissue)

For Immunohistochemistry:

• Known expression pattern controls (e.g., brain regions with established CHRNA7 expression)

• Serial dilution controls to determine optimal antibody concentration

• Antigen retrieval controls (with and without retrieval)

For Human Samples:

• CHRNA7/CHRFAM7A differentiation controls

• qRT-PCR with specific primers for each gene

• Functional assays to distinguish receptor populations

The Moser et al. study (2015) provides an excellent model for comprehensive antibody validation using knockout tissues, multiple applications (IHC, WB), qRT-PCR, and 2D gel electrophoresis with mass spectrometry .

How is CHRNA7 involved in inflammatory responses, and what methodological approaches best investigate this function?

CHRNA7 plays a crucial role in regulating inflammatory responses through the cholinergic anti-inflammatory pathway:

Mechanistic understanding:

• CHRNA7 activation suppresses LPS-induced TNF-α release in macrophages and microglia

• Vagus nerve stimulation can suppress inflammation through CHRNA7 activation

• In humans, the CHRFAM7A/CHRNA7 ratio increases during inflammatory responses

Methodological approaches for investigating CHRNA7 in inflammation:

Experimental models:

• Human macrophage cultures with LPS stimulation to assess CHRFAM7A/CHRNA7 ratio changes

• Comparison between human and animal models to identify species-specific responses

• Patient-derived samples to correlate inflammatory markers with receptor expression

Emerging research directions:

• The association between CHRNA7/CHRFAM7A ratio and psychiatric disorders (schizophrenia, bipolar disorder)

• Role of α7-nAChR autoantibodies in blocking receptor function similar to NMDA-R autoantibodies in encephalitis

• Potential for CHRNA7 agonists as anti-inflammatory therapeutics

This research area highlights the complex interplay between the nervous and immune systems, with CHRNA7 serving as a key mediator with human-specific regulatory mechanisms.

What are the most reliable methods for quantifying CHRNA7 expression levels across different experimental systems?

Quantifying CHRNA7 expression requires different approaches depending on whether protein or RNA levels are being measured:

Protein Quantification Methods:

RNA Quantification Methods:

Integrated approaches for highest reliability:

• Combine protein and RNA detection methods

• Use multiple antibodies targeting different epitopes

• Include functional assays (electrophysiology, calcium imaging)

• Validate in systems with manipulated expression (overexpression, knockdown)

• Account for CHRFAM7A expression in human samples

The CHRFAM7A/CHRNA7 ratio is particularly important in human studies and can be measured precisely with qRT-PCR using specific primers for each gene, serving as a meaningful biomarker for inflammatory states .

How can researchers address the challenge of antibody cross-reactivity when studying CHRNA7?

Addressing CHRNA7 antibody cross-reactivity requires systematic approaches:

Identified cross-reactivity issues:

• Cross-reactivity with CHRFAM7A in human samples

• Documented cross-reactivity with β-actin and β-enolase

• Potential cross-reactivity with other nicotinic receptor subunits

• Non-specific binding in various tissues

Comprehensive validation strategy:

Human-specific considerations:

• For human samples, design experiments to explicitly distinguish CHRNA7 and CHRFAM7A

• Consider calculating CHRFAM7A/CHRNA7 ratio by qRT-PCR in parallel with protein studies

• When possible, include functional assays to distinguish receptors

Experimental reporting recommendations:

• Document all antibody validation steps in publications

• Report catalog numbers, dilutions, and specific protocols

• Include representative images of both positive and negative controls

• Acknowledge potential cross-reactivity limitations

The comprehensive approach demonstrated by Moser et al. (2015) provides an excellent model for antibody validation, showing how rigorous testing revealed significant specificity issues with commonly used antibodies .