Cnpy2 Antibody

What is CNPY2 and why is it important in neurological research?

CNPY2 is an endoplasmic reticulum (ER)-localized protein belonging to the CNPY gene family. It has gained significant research interest due to its protective role against ER stress in neuronal cells, particularly in the context of neurodegenerative diseases. CNPY2 reduces the activating transcription factor 6 (ATF6) branch of ER stress and decreases the expression of CCAT/Enhancer-Binding Protein Homologous Protein (CHOP) involved in cell death .

Immunostaining studies have revealed that CNPY2 is expressed by cortical and striatal neurons and is co-expressed with the transcription factor COUPTF-interacting protein 2 (CTIP2), also called B-cell lymphoma/leukemia 11 B (BCL11B) . This expression pattern in the corticostriatal circuitry, which regulates body movements, makes CNPY2 particularly relevant for research on movement disorders like Huntington's disease .

What are the optimal applications for CNPY2 antibodies in neuroscience research?

CNPY2 antibodies have been validated for multiple applications in neuroscience research:

For neuronal tissue analysis, researchers commonly use antigen retrieval by heating sections in 10 mM citrate buffer (pH 6.0) with 0.05% Tween-20 for 10 minutes, followed by rinsing with PBS and blocking using 1% BSA, 0.2% Triton-X-100 in PBS for 1 hour before applying the primary antibody . This protocol enhances antibody binding in fixed brain tissues where epitopes may be masked.

How can I validate the specificity of my CNPY2 antibody?

Proper validation ensures reliable experimental results. Follow these methodological steps:

• Recombinant protein testing: Express CNPY2-GFP and CNPY2-V5-His constructs in HEK293 cells after transient transfection and perform immunoblotting to confirm antibody recognition of tagged proteins at expected molecular weights .

• Multiple detection methods: Compare results from different applications (WB, IHC, ICC/IF) to ensure consistent detection patterns.

• Positive controls: Include samples known to express CNPY2, such as neuroblastoma cell lines treated with tunicamycin, which increases CNPY2 expression .

• Immunoprecipitation validation: Perform immunoprecipitation followed by immunoblotting to confirm specificity, especially useful for detecting secreted forms of CNPY2 .

• Knockout/knockdown controls: Use CNPY2 shRNA expression systems to create negative controls by downregulating CNPY2 expression .

Why do CNPY2 antibodies detect multiple molecular weight bands, and what does this mean for my research?

CNPY2 appears as two distinct bands in immunoblots with molecular weights of approximately 21 kDa and 17 kDa . This pattern has important implications:

• The 21 kDa band represents the full-length CNPY2 protein

• The 17 kDa band likely represents the mature form of CNPY2 following removal of the signal sequence

• The 17 kDa CNPY2 form has been detected in cell culture medium, suggesting it is secreted

When analyzing experimental results, track both forms separately as they may have different functions or localizations. In N171-82Q mouse models of Huntington's disease, the 21 kDa CNPY2 band showed more pronounced changes than the 17 kDa band in both striatum and cortex . Ensure your experimental design includes controls that allow differentiation between these forms, especially when studying secretion versus intracellular retention of CNPY2.

What is the optimal protocol for detecting CNPY2 in mouse brain sections?

For optimal detection of CNPY2 in mouse brain sections, follow this validated protocol:

• Tissue preparation: Perfuse mice with 4% paraformaldehyde and post-fix tissues before processing for paraffin or frozen sections.

• Antigen retrieval: Heat sections in 10 mM citrate buffer (pH 6.0) with 0.05% Tween-20 for 10 minutes.

• Blocking: Rinse with PBS, then block using 1% BSA, 0.2% Triton-X-100 in PBS for 1 hour.

• Primary antibody incubation: Apply rabbit polyclonal anti-CNPY2 antibody (diluted 1:250) along with other primary antibodies of interest (e.g., rat anti-CTIP2 diluted 1:200) and incubate overnight at 4°C .

• Secondary antibody and visualization: Apply appropriate fluorescent secondary antibodies and counterstain nuclei if desired.

• Mounting and imaging: Mount slices using appropriate medium (e.g., DPX) and generate images using a digital slide scanner or fluorescence microscope .

• Quantification: Use ImageJ software to count the number of CNPY2 and CTIP2 immunopositive cells from multiple fields per region .

How do CNPY2 expression patterns change in neurodegenerative disease models?

In the N171-82Q mouse model of Huntington's disease, CNPY2 expression shows region-specific and age-dependent changes:

These findings suggest region-specific vulnerability to ER stress in HD pathogenesis . When designing experiments to study CNPY2 in disease models, consider:

• Including multiple age points to capture dynamic changes

• Examining both cortical and striatal regions

• Correlating CNPY2 expression with markers of ER stress

• Quantifying both immunohistochemical staining and protein levels via immunoblotting

This approach provides a more comprehensive understanding of how CNPY2 expression correlates with disease progression .

What is the relationship between CNPY2 and ER stress pathways in neurons?

CNPY2 has a complex relationship with ER stress pathways in neurons:

• CNPY2 expression is regulated by ER stress: Treatment with tunicamycin (an ER stress inducer) increases CNPY2 expression and protein levels in neuroblastoma cells, with peak protein levels at 4 hours post-treatment .

• CNPY2 interacts with PERK: CNPY2 co-immunoprecipitates with PERK and directly binds to the PERK luminal domain (PERK-LD), as confirmed by pulldown experiments and isothermal titration calorimetry .

• CNPY2 is a CHOP target gene: The CNPY2 gene contains a CHOP consensus recognition motif in its promoter, and CHOP binding to this promoter increases following tunicamycin treatment .

• CNPY2 provides protection against ER stress: Overexpression of CNPY2 enhances neuroblastoma cell viability after tunicamycin treatment, while CNPY2 downregulation reduces viability .

• CNPY2 modulates UPR pathways: CNPY2 reduces the ATF6 branch of ER stress and decreases CHOP expression, potentially creating a negative feedback loop in the UPR .

When investigating these pathways, consider using multiple ER stress inducers (both tunicamycin and thapsigargin have been shown to increase CNPY2 levels) and examining time-dependent changes in CNPY2 expression relative to other UPR components .

How can I optimize co-immunostaining for CNPY2 and CTIP2 in brain sections?

For successful co-immunostaining of CNPY2 and CTIP2 in brain sections, follow these optimization steps:

• Antibody selection: Use rabbit polyclonal anti-CNPY2 (such as Proteintech 14,635-1-AP, diluted 1:250) and rat anti-CTIP2 (such as Abcam ab18465, diluted 1:200) . These antibodies have been validated for co-staining.

• Antigen retrieval optimization: Adequate heat-induced epitope retrieval is critical. Test different citrate buffer concentrations and heating times if initial results are suboptimal.

• Sequential antibody application: If cross-reactivity occurs, consider sequential rather than simultaneous antibody incubation.

• Species-specific secondary antibodies: Use highly cross-adsorbed secondary antibodies with minimal species cross-reactivity.

• Control for fluorophore bleed-through: Include single-stained controls to ensure fluorescence channel separation.

• Quantification method: For cell counting, use ImageJ software on fluorescence images captured at 60x magnification from specific brain regions (e.g., cortical layers 5/6 and upper striatum) .

This approach has successfully demonstrated co-expression of CNPY2 and CTIP2 in corticostriatal neurons, providing insights into the role of CNPY2 in movement-related neural circuits .

What are the technical considerations for detecting secreted versus intracellular CNPY2?

Detecting secreted versus intracellular CNPY2 requires different methodological approaches:

For secreted CNPY2 detection:

• Collect cell culture media from experimental and control conditions.

• Pre-clear the medium using Protein A/G agarose beads (1 hour at 4°C).

• Incubate pre-cleared suspension with anti-CNPY2 antibody (2 μg) overnight at 4°C.

• Add agarose beads and rotate for 2-3 hours at 4°C.

• Centrifuge at 9000g for 1 minute at 4°C and wash three times.

• Resuspend in 2x Laemmli buffer, heat at 95°C for 5 minutes, and analyze by immunoblotting .

This approach successfully detected the 17 kDa CNPY2 form in cell culture medium, with a slight increase observed 7 hours after tunicamycin treatment .

How should I approach CNPY2 antibody selection for different experimental purposes?

Select CNPY2 antibodies based on your specific experimental needs:

For optimal results:

• Verify the immunogen sequence: For instance, Sigma's HPA038466 targets the sequence "RRSQDLHCGACRALVDELEWEIAQVDPKKTIQMGSFRINPDGSQSVVEVPYARSEAHLTELLEEICDRMKEYGEQIDPSTHRKNYVRVVGRNGESSELDLQGIRIDSD" , while Biomatik's CAU21302 targets Arg21~Leu182 .

• Check experimental validation: Review available validation data for your application of interest.

• Consider epitope accessibility: For detecting specific forms of CNPY2 (secreted vs. membrane-bound), select antibodies targeting accessible epitopes in that configuration.

• Test multiple antibodies when possible: Different antibodies may provide complementary information about CNPY2 expression and localization.