CPNE4 Antibody

What is CPNE4 and why is it significant in neuroscience research?

CPNE4 (Copine-4) is a calcium-dependent phospholipid-binding protein belonging to the evolutionarily conserved copine family. It has significant neurological implications as it contains two C2 domains (C2A and C2B) and a von Willebrand Factor A (vWA) domain . The C2 domains facilitate calcium-mediated membrane attachment while the vWA domain mediates protein-protein interactions . CPNE4 is uniquely expressed in Retinal Ganglion Cells (RGCs) with minor expression in one amacrine cell type in the Inner Nuclear Layer, making it particularly valuable for retinal research . Research indicates CPNE4 likely participates in synaptic plasticity due to its membrane-binding capabilities and interactions with synaptic proteins .

What types of CPNE4 antibodies are commercially available for research purposes?

Currently, the majority of commercially available CPNE4 antibodies are rabbit polyclonal antibodies that target different epitopes of the protein . These antibodies are generated using various immunogens:

Research groups have also developed custom antibodies targeting specific epitopes for specialized studies .

What is the predicted versus observed molecular weight of CPNE4 in western blot analysis?

• Commercial antibodies typically detect CPNE4 at the expected 62 kDa

• Some studies have reported higher molecular weight bands (~175 kDa)

This discrepancy between predicted and observed weights may result from:

• Post-translational modifications

• Protein-protein interactions that are not fully disrupted during sample preparation

• Splice variants of CPNE4

• Different denaturing conditions used in sample preparation

When performing western blot analysis, it's advisable to include appropriate positive controls such as mouse or rat brain tissue lysates where CPNE4 is known to be expressed .

What are the optimal protocols for CPNE4 antibody application in Western Blot analysis?

Based on multiple research protocols, the following represents an optimized Western Blot procedure for CPNE4 detection:

• Sample preparation:

  • Fresh or frozen brain tissue (particularly from mouse or rat) provides reliable positive controls

  • Lyse tissue in buffer containing protease inhibitors, 5mM DTT, 5mM EGTA, and 1mM PMSF

  • Sonicate and centrifuge at 13,000 rpm for 30 minutes at 4°C

• Electrophoresis and transfer:

  • Separate 20-50 μg of protein on a 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

• Antibody incubation:

  • Block membrane in 5% milk in TBST for 1 hour at room temperature

  • Incubate with primary CPNE4 antibody at recommended dilution (typically 1:500-1:3000) overnight at 4°C

  • Wash 3× with TBST

  • Incubate with appropriate secondary antibody (goat anti-rabbit IgG HRP conjugate at 1:5000-1:50000) for 1 hour at room temperature

  • Wash 3× with TBST

• Detection:

  • Apply chemiluminescence substrate and image using standard techniques

  • Expected band size: 62 kDa (occasional higher molecular weight bands may be observed)

Troubleshooting: If non-specific bands appear, further optimization of antibody concentration or more stringent washing conditions may be required.

How should researchers optimize immunohistochemistry protocols for CPNE4 detection in neural tissues?

For optimal CPNE4 immunohistochemistry in neural tissues, particularly retinal sections:

• Tissue preparation:

  • Fix freshly dissected tissue in 4% paraformaldehyde for 1-2 hours at room temperature

  • For retinal tissue: consider extending fixation to ensure proper penetration

  • Cryoprotect in 30% sucrose and embed in OCT compound

  • Section at 12-16 μm thickness for optimal antibody penetration

• Staining protocol:

  • Antigen retrieval: Citrate buffer (pH 6.0) at 95°C for 20 minutes is recommended for paraffin sections

  • Block with 5-10% normal serum (matching the species of secondary antibody) and 0.3% Triton X-100

  • Incubate with primary CPNE4 antibody at appropriate dilution:

    • Commercial antibodies: 1:100 for IHC-P

    • Custom C-terminal antibody: 1:2000

    • Custom N-terminal antibody: 1:400

  • Incubate overnight at 4°C

  • Wash in PBS (3× 10 minutes)

  • Apply appropriate secondary antibodies (fluorescent or HRP-conjugated)

  • For fluorescent detection, counterstain with DAPI to visualize nuclei

• Controls and validation:

  • Include negative controls (omitting primary antibody)

  • Use tissue from CPNE4 knockout models if available

  • Consider co-staining with RGC markers (e.g., Brn3a, Brn3b) as CPNE4 is specifically expressed in RGCs

Notable findings: CPNE4 is primarily localized in the Ganglion Cell Layer (GCL) of the retina, with subcellular localization in cell bodies and dendrites, rarely reaching into axons .

What validation procedures should be used to confirm CPNE4 antibody specificity?

To ensure experimental validity, comprehensive validation of CPNE4 antibody specificity is crucial:

• Genetic validation approaches:

  • Testing on CPNE4 knockout tissue/cells (gold standard)

  • siRNA/shRNA knockdown of CPNE4 followed by antibody testing

  • Overexpression of tagged CPNE4 constructs and co-localization with antibody staining

• Biochemical validation:

  • Pre-absorption test: Pre-incubate antibody with excess immunizing peptide before use

  • Cross-reactivity testing against other copine family members (particularly CPNE5, CPNE6, CPNE8)

  • Confirm the detection of recombinant CPNE4 at predicted molecular weight

• Orthogonal validation:

  • Compare results between antibodies targeting different epitopes of CPNE4

  • Compare protein detection results with mRNA expression data

  • Mass spectrometry validation of immunoprecipitated proteins

• Controls to include:

  • Positive control tissues (brain, particularly retina)

  • Negative control (tissue with minimal CPNE4 expression)

  • Omission of primary antibody

  • Isotype control antibody

Researchers should be aware that many commercially available antibodies lack comprehensive validation, contributing to the "antibody characterization crisis" discussed in the literature . This emphasizes the importance of conducting validation experiments within your specific experimental system.

How can CPNE4 antibodies be effectively used to investigate protein-protein interactions?

CPNE4 protein interactions are mediated primarily through its vWA domain. The following methodologies can be employed to study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in a buffer containing 5mM DTT, 5mM EGTA, 1mM PMSF in PBS with 0.5% Triton X-100

  • Immunoprecipitate with CPNE4 antibody bound to protein A/G beads

  • Wash extensively and elute proteins

  • Analyze by Western blotting with antibodies against suspected interacting proteins

  Research has identified several CPNE4 interacting proteins using this approach, including Morn2, HCFC1, and Mycbp2 .

• Yeast two-hybrid (Y2H) analysis:

  • Use the CPNE4 vWA domain as bait protein

  • Screen against a cDNA library (retinal cDNA library provides tissue-specific interactions)

  • Validate positive interactions through sequential screening and sequencing

  Previous Y2H screens have identified interactions between CPNE4's vWA domain and proteins such as Morn2, HCFC1, and Tox3 .

• Pull-down assays with GST-fusion proteins:

  • Express GST-vWA or GST-CPNE4 fusion proteins in bacterial systems

  • Incubate with retinal protein extracts

  • Analyze bound proteins by LC-MS

  This approach has been successfully used to identify native CPNE4 binding partners in retinal tissue .

• Proximity labeling approaches:

  • Express CPNE4 fused to BioID or APEX2

  • Identify proteins in close proximity through biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

When interpreting results, consider that different methodologies may capture different types of interactions (stable vs. transient, direct vs. indirect).

What approaches should be used to study CPNE4 subcellular localization in neuronal cells?

CPNE4 demonstrates complex subcellular localization patterns in neuronal cells. The following approaches provide comprehensive analysis:

• High-resolution confocal microscopy:

  • Use Airyscan or similar super-resolution techniques for detailed visualization

  • Prepare primary neuronal cultures or thin tissue sections (≤15 μm)

  • Apply CPNE4 antibody at 1:100-1:200 dilution

  • Co-stain with markers for subcellular compartments:

    • Membrane markers (e.g., membrane-tagged GFP)

    • Dendritic markers (e.g., MAP2)

    • Axonal markers (e.g., Tau)

    • Synaptic markers (e.g., PSD95, synaptophysin)

• Live cell imaging with fluorescent protein fusions:

  • Generate full-length CPNE4-GFP/RFP fusion constructs

  • Express in primary neurons or neuronal cell lines

  • Monitor dynamic localization in response to stimuli (particularly calcium influx)

• Subcellular fractionation and Western blotting:

  • Prepare cytosolic, membrane, nuclear, and synaptosomal fractions

  • Analyze CPNE4 distribution by Western blotting

  • Include fraction-specific markers as controls

Research findings indicate that CPNE4 localizes to:

• Cell bodies and dendrites of RGCs, rarely reaching into axons

• Nuclei, cell body, and plasma membrane when expressed in cell lines

• Large varicosities or "blebs" on dendrites when overexpressed in RGCs

The calcium-dependent nature of CPNE4 membrane association should be considered when designing experiments, as calcium levels may significantly affect localization patterns.

How can researchers use CPNE4 antibodies to investigate its role in morphological development of neurons?

CPNE4 has been implicated in neuronal morphology development, particularly in RGCs. The following methodologies can help elucidate its function:

When interpreting results, consider potential compensatory mechanisms from other copine family members, particularly those expressed in neurons (CPNE5, CPNE6, CPNE8, CPNE9).

What are the common technical challenges when using CPNE4 antibodies and how can they be addressed?

Researchers working with CPNE4 antibodies may encounter several technical challenges:

• Inconsistent band sizes in Western blots:

  • Challenge: Detection of unexpected molecular weight bands (e.g., 175 kDa instead of predicted 62 kDa)

  • Solutions:

    • Include positive controls (brain tissue lysates)

    • Optimize sample preparation (reduce protein aggregation with stronger denaturing conditions)

    • Test multiple antibodies targeting different epitopes

    • Confirm specificity using knockout/knockdown controls

• Cross-reactivity with other copine family members:

  • Challenge: Potential detection of related proteins (CPNE5, CPNE8, etc.) due to sequence homology

  • Solutions:

    • Validate antibody against recombinant copine proteins

    • Include appropriate controls (tissues from CPNE4 knockout models)

    • Use specific peptide competition assays

    • Compare staining patterns with mRNA expression data

• Variable immunostaining results:

  • Challenge: Inconsistent staining intensity or patterns between experiments

  • Solutions:

    • Optimize fixation conditions (duration, fixative composition)

    • Test multiple antigen retrieval methods

    • Titrate antibody concentration carefully

    • Process control and experimental samples simultaneously

    • Consider tissue penetration issues (reduce section thickness)

• Limited antibody availability for certain applications:

  • Challenge: Not all commercially available antibodies work in all applications

  • Solutions:

    • Refer to manufacturer validation data and application recommendations

    • Test multiple commercial antibodies

    • Consider generating custom antibodies for specific applications

    • Collaborate with labs that have validated antibodies for your application

• Storage and handling issues:

  • Challenge: Antibody degradation leading to reduced sensitivity

  • Solutions:

    • Store according to manufacturer recommendations (typically -20°C in small aliquots)

    • Avoid repeated freeze-thaw cycles

    • Add preservatives if storing diluted antibody (0.02% sodium azide)

    • Check expiration dates and antibody appearance before use

How can researchers resolve discrepancies in experimental results using different CPNE4 antibodies?

When different CPNE4 antibodies yield inconsistent results, systematic troubleshooting is essential:

• Epitope mapping and comparison:

  • Identify the exact epitopes recognized by each antibody

  • Consider whether post-translational modifications might affect epitope accessibility

  • Evaluate whether different splice variants might be detected differentially

  • Test antibodies targeting different regions (N-terminal vs. C-terminal)

• Standardized validation approach:

  • Test all antibodies simultaneously on the same samples

  • Include positive and negative controls for each antibody

  • Document all experimental conditions meticulously

  • Quantify results using standardized metrics where possible

• Orthogonal method validation:

  • Verify findings using complementary techniques (e.g., if IHC results differ, compare with in situ hybridization)

  • Consider RNAscope or FISH to confirm mRNA expression patterns

  • Use tagged recombinant CPNE4 expression as a control

  • Apply mass spectrometry to confirm protein identity

• Controls to resolve discrepancies:

  • Use CPNE4 knockout tissues as definitive negative controls

  • Include peptide competition assays for each antibody

  • Test on overexpression systems with known CPNE4 content

  • Consider using a third antibody as a tiebreaker

• Documentation and reporting:

  • Clearly document antibody sources, catalog numbers, and lot numbers

  • Report detailed methodological conditions when publishing

  • Acknowledge limitations in interpretation when discrepancies remain unresolved

  • Consider the antibody characterization crisis context when interpreting results

Remember that antibodies represent different tools that may excel in different applications. A single antibody may not be optimal for all experimental conditions.

What criteria should be considered when selecting a CPNE4 antibody for specific research applications?

Selection of the appropriate CPNE4 antibody should be guided by several critical factors:

• Application compatibility:

  • Verify validation data for your specific application (WB, IHC, IF, IP, ELISA)

  • Check recommended dilutions for each application

  • Some antibodies may work well for WB but poorly for IHC, or vice versa

  • Application table example:

  Application	Recommended Antibody Features	Typical Dilutions
  Western Blot	High specificity, validated in tissue lysates	1:500-1:3000
  IHC-P	Optimized for fixed tissues, validated in brain/retina	1:20-1:200
  IF/ICC	High signal-to-noise ratio, minimal background	1:50-1:800
  IP	High affinity, validated for pull-down	Application-specific

• Epitope characteristics:

  • Consider whether the epitope is conserved across species of interest

  • Evaluate accessibility of the epitope in native vs. denatured protein

  • N-terminal antibodies may detect different splice variants than C-terminal ones

  • Review the immunogen sequence and compare to your research model

• Validation evidence:

  • Assess the rigor of validation data provided

  • Look for knockout/knockdown validation

  • Check for cross-reactivity testing with other copine family members

  • Consider independent validation studies in the literature

• Species reactivity:

  • Confirm reactivity with your experimental species (human, mouse, rat, etc.)

  • Check sequence homology between species for the specific epitope

  • Be cautious of predicted reactivity without experimental validation

• Technical specifications:

  • Antibody format (unconjugated, conjugated to HRP, fluorophores, biotin)

  • Clone type (monoclonal vs. polyclonal)

  • Production host (to avoid cross-reactivity in multi-labeling experiments)

  • Storage requirements and stability

• Research context:

  • For studies focusing on protein-protein interactions, antibodies validated for IP may be preferable

  • For developmental studies, antibodies that work in embryonic tissues should be prioritized

  • For quantitative applications, consider antibodies with established linear detection range

The ideal approach is to test multiple antibodies in parallel for your specific application and experimental system before committing to a large-scale study.

How are CPNE4 antibodies being used to understand the role of CPNE4 in retinal ganglion cell development and function?

CPNE4 antibodies are instrumental in uncovering the unique role of CPNE4 in RGC biology:

• Developmental expression profiling:

  • CPNE4 antibodies have revealed that, unlike other copine family members, CPNE4 is specifically expressed in RGCs with minor expression in one amacrine cell type

  • Temporal expression analysis during retinal development helps correlate CPNE4 expression with key developmental milestones

• Subcellular localization studies:

  • High-resolution imaging with CPNE4 antibodies has shown that the protein localizes primarily to RGC cell bodies and dendrites, rarely reaching into axons

  • This distribution pattern suggests a role in dendritic development or synaptic function rather than axonal processes

• Functional studies through overexpression:

  • When combined with morphological analysis, CPNE4 antibodies have demonstrated that overexpression of CPNE4 in RGCs induces formation of large dendritic varicosities ("blebs")

  • This phenotype suggests a role in membrane trafficking or organelle distribution within dendrites

• Regulation by transcription factors:

  • CPNE4 expression is regulated by the Brn3b and Brn3a transcription factors

  • Co-labeling with Brn3 and CPNE4 antibodies helps establish regulatory relationships in developing RGCs

• Protein-protein interaction networks:

  • CPNE4 antibodies facilitate immunoprecipitation experiments that, when combined with mass spectrometry, have identified interacting partners including Morn2, HCFC1, and Mycbp2

  • These interactions provide insights into molecular pathways involving CPNE4 in retinal function

Current research suggests CPNE4 may function in membrane trafficking and protein complex assembly, potentially contributing to dendritic morphogenesis and synaptic organization in RGCs.

What role might CPNE4 antibodies play in investigating neurodegenerative diseases affecting retinal ganglion cells?

Given CPNE4's specific expression in RGCs, antibodies against this protein have potential applications in neurodegenerative disease research:

• RGC-specific marker in degenerative conditions:

  • CPNE4 antibodies can serve as selective markers for RGCs in models of glaucoma, optic neuropathies, and other conditions where RGC degeneration occurs

  • Quantification of CPNE4-positive cells provides a measure of RGC survival

• Pathological changes in CPNE4 expression or localization:

  • CPNE4 antibodies can reveal alterations in expression levels or subcellular distribution during disease progression

  • Changes in CPNE4 localization might serve as early indicators of RGC stress before morphological degeneration becomes apparent

• Interaction with disease-associated proteins:

  • Co-immunoprecipitation using CPNE4 antibodies could identify altered protein interactions in disease states

  • Mass spectrometry analysis of CPNE4 immunoprecipitates from healthy versus diseased tissue might reveal disease-specific interaction partners

• Calcium signaling dysregulation:

  • As a calcium-dependent protein, CPNE4 function may be altered in conditions with disrupted calcium homeostasis

  • CPNE4 antibodies can help track the protein's response to calcium dysregulation in degenerative models

• Therapeutic target validation:

  • For potential therapies targeting CPNE4 or its pathways, antibodies provide essential tools for target engagement studies

  • Phospho-specific antibodies (if developed) could monitor activation states in response to therapeutic interventions

Research into CPNE4's role in RGC degeneration is still emerging, but its specific expression pattern makes it a promising candidate for studies of RGC-specific pathologies.

How can researchers utilize CPNE4 antibodies in combination with advanced imaging techniques to gain new insights into neuronal membrane dynamics?

Integration of CPNE4 antibodies with cutting-edge imaging approaches offers powerful avenues for investigating neuronal membrane processes:

• Super-resolution microscopy:

  • Techniques such as STED, STORM, or PALM combined with CPNE4 immunolabeling can reveal nanoscale distribution patterns

  • Resolution of CPNE4 clustering at specific membrane microdomains may provide functional insights

  • Multi-color super-resolution with synaptic markers can precisely map CPNE4's relationship to synaptic structures

• Live-cell calcium imaging coupled with fixed-cell CPNE4 immunolabeling:

  • Record calcium dynamics in living neurons

  • Fix and immunolabel for CPNE4 to correlate calcium activity patterns with CPNE4 distribution

  • This correlative approach can link functional calcium signals to CPNE4 localization

• Expansion microscopy:

  • Physical expansion of specimens followed by CPNE4 immunolabeling allows visualization of fine structures using standard confocal microscopy

  • Particularly valuable for studying CPNE4 distribution within dendritic spines or other small neuronal compartments

• FRAP (Fluorescence Recovery After Photobleaching) with antibody validation:

  • Express fluorescently-tagged CPNE4 and perform FRAP to measure mobility

  • Validate dynamics using antibodies against endogenous CPNE4

  • This approach can reveal the mobile versus stable pools of CPNE4 at membranes

• Cryo-electron microscopy with immunogold labeling:

  • Ultrastructural localization of CPNE4 at membranes and vesicular structures

  • Potential visualization of CPNE4 in relation to membrane curvature or specialized membrane domains

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescent CPNE4 antibody labeling with electron microscopy of the same sample

  • This approach links functional distribution patterns with ultrastructural features

These advanced imaging approaches, when combined with appropriate CPNE4 antibodies, can provide unprecedented insights into how this calcium-dependent protein participates in membrane dynamics, potentially revealing its role in membrane trafficking and synaptic plasticity .