CPNE2 Antibody

What is CPNE2 and what cellular processes does it participate in?

CPNE2 (Copine-2) is a calcium-dependent phospholipid-binding protein that functions in various cellular processes. It contains two Ca²⁺-dependent phospholipid-binding domains known as C2 domains, which are critical for its function . CPNE2 plays significant roles in:

• Calcium signaling pathways, where it acts as a calcium sensor

• Membrane trafficking mechanisms between cellular compartments

• Protein-protein interactions and associated signaling cascades

• Potentially in cellular homeostasis regulation

Understanding these functions is essential when designing experiments to investigate CPNE2's role in normal physiology or disease states. When studying CPNE2, consider that its calcium-binding properties may necessitate careful buffer composition in your experimental protocols.

What applications are CPNE2 antibodies validated for?

Current commercial CPNE2 antibodies have been validated for several common research applications:

When planning experiments, consider that different applications require distinct antibody characteristics. For example, antibodies that perform well in Western blot may not necessarily be optimal for immunohistochemistry due to differences in epitope accessibility and fixation effects. Always validate antibodies for your specific application and experimental conditions, even if previously validated for similar purposes.

What tissues or cell lines are recommended for CPNE2 expression studies?

Based on available data, the following tissues and cell lines show notable CPNE2 expression:

• Brain tissue (particularly useful for both human and mouse studies)

• HEK-293, HeLa, and U2OS cell lines have been used in general protein expression studies, though specific CPNE2 expression levels in these lines should be verified

When selecting cell lines for CPNE2 research, consider:

• Consulting proteomic databases like PaxDb (https://pax-db.org/) to identify high-expressing cell lines

• Performing preliminary Western blot analysis to confirm expression in your chosen model

• Including appropriate positive control tissues (such as brain tissue) in your experimental design

• Avoiding assumptions about tissue-specific expression patterns, as many proteins are expressed more broadly than traditionally thought

How should CPNE2 antibodies be stored to maintain their efficacy?

Proper storage of CPNE2 antibodies is critical for maintaining their specificity and sensitivity:

• Store at -20°C for long-term preservation

• Most CPNE2 antibodies are supplied in buffer solutions containing stabilizers:

  • Typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some may contain 0.1% BSA as an additional stabilizer

• Antibodies are generally stable for one year after shipment when stored properly

• For PACO38718, the recommended storage buffer is 0.03% Proclin 300 in 50% Glycerol, 0.01M PBS, pH 7.4

Best practices for antibody handling include:

• Avoiding repeated freeze-thaw cycles by preparing small aliquots upon receipt

• Allowing antibodies to reach room temperature before opening to prevent condensation

• Brief centrifugation of the vial before opening to collect all liquid

• Maintaining sterile conditions when handling

What validation strategies should be employed to confirm CPNE2 antibody specificity?

A rigorous validation approach for CPNE2 antibodies should include:

• CRISPR/Cas9 knockout controls: Generate CPNE2 knockout cell lines in relevant models to serve as negative controls. This approach represents the gold standard for antibody validation .

• Multi-technique validation sequence:

  • Begin with Western blot in high-expressing cell lines

  • Progress to immunoprecipitation validation

  • Confirm results with immunofluorescence studies

  • Finally test in more complex applications like immunohistochemistry

• Expression system validation: Test the antibody against recombinant CPNE2 expressed in a controlled system at various concentrations

• Peptide competition assays: Pre-incubate the antibody with purified CPNE2 peptide to confirm that signal disappearance results from specific binding

• Cross-reactivity assessment: Particularly important for polyclonal antibodies like PACO38718 and 17664-1-AP, test against closely related family members (other copines) to confirm specificity

This comprehensive validation approach helps prevent misinterpretation of results due to antibody non-specificity, which has been noted as a significant issue in the research community .

How can I optimize antigen retrieval for CPNE2 immunohistochemistry?

Effective antigen retrieval is critical for CPNE2 detection in fixed tissues:

For antibody 17664-1-AP, specific recommendations include:

• Primary recommendation: TE buffer at pH 9.0

• Alternative approach: Citrate buffer at pH 6.0

General optimization strategy for CPNE2 antibodies:

• Comparative method testing:

  • Heat-induced epitope retrieval (HIER) with various buffers (citrate, EDTA, Tris)

  • Enzymatic retrieval (proteinase K, trypsin)

  • pH variations (acidic vs. alkaline conditions)

• Time and temperature optimization:

  • Test different durations (10-30 minutes)

  • Compare various heating methods (microwave, pressure cooker, water bath)

• Tissue-specific considerations:

  • Brain tissue may require more gentle retrieval to preserve morphology

  • Consider tissue thickness and fixation conditions

• Validation approach:

  • Include known positive control tissues (brain tissue for CPNE2)

  • Run parallel sections with and without retrieval

  • Compare retrieval methods side-by-side on serial sections

Document all parameters systematically to establish a reproducible protocol for your specific experimental conditions.

What are the methodological considerations for using CPNE2 antibodies in co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments to investigate CPNE2 protein interactions:

• Buffer optimization:

  • Consider CPNE2's calcium-dependent properties - buffers should either preserve calcium binding (include Ca²⁺) or deliberately disrupt it (include EDTA/EGTA) depending on experimental goals

  • Test multiple lysis conditions (RIPA vs. gentler NP-40 or digitonin buffers)

  • Include appropriate protease inhibitors to prevent degradation

• Antibody selection considerations:

  • For CPNE2 pull-down, polyclonal antibodies may offer advantages due to recognition of multiple epitopes

  • Consider using multiple antibodies recognizing different epitopes to confirm results

  • Verify that the antibody does not interfere with protein interaction interfaces

• Controls and validation:

  • Include IgG isotype controls

  • Perform reciprocal co-IPs when possible

  • Validate with recombinant CPNE2 protein

  • Consider comparing results in calcium-depleted vs. calcium-rich conditions

• Detection strategy:

  • When probing for co-precipitated proteins, optimize antibody dilutions for Western blot:

    • For 17664-1-AP: 1:500-1:1000

  • Consider using HRP-conjugated light-chain specific secondary antibodies to avoid heavy chain interference

Given CPNE2's role in protein-protein interactions and membrane trafficking, co-IP represents a valuable approach for identifying its binding partners and regulatory mechanisms .

How can I effectively troubleshoot non-specific binding or weak signals with CPNE2 antibodies?

Common challenges with CPNE2 antibodies can be systematically addressed through these approaches:

For non-specific binding:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, normal serum)

  • Increase blocking duration or concentration

  • Consider specialized blocking reagents for specific tissues

• Titrate antibody concentrations:

  • For PACO38718: Test dilutions between 1:20-1:200 for IHC

  • For 17664-1-AP: Test dilutions between 1:50-1:500 for IHC

  • Systematically evaluate signal-to-noise ratio at each dilution

• Increase washing stringency:

  • Extend washing times

  • Add detergents (0.1-0.3% Tween-20)

  • Increase salt concentration in wash buffers

For weak signals:

• Signal amplification strategies:

  • Employ tyramide signal amplification (TSA)

  • Use biotin-streptavidin systems

  • Consider more sensitive detection systems (SuperSignal West Femto vs. ECL)

• Sample preparation optimization:

  • For brain tissue, optimize fixation time

  • Adjust antigen retrieval conditions (as detailed in FAQ 2.2)

  • Consider fresh vs. frozen tissue preparation methods

• Antibody-specific considerations:

  • Some antibodies may perform better in specific applications

  • PACO38718 shows high specificity for IHC in human samples

  • 17664-1-AP has been validated for both mouse and human samples

Systematic troubleshooting approach:

• Change only one variable at a time

• Document all modifications

• Include appropriate positive and negative controls

• Consider testing multiple antibodies targeting different CPNE2 epitopes

What methodologies are recommended for studying CPNE2's calcium-dependent functions?

CPNE2's calcium-dependent phospholipid-binding properties require specialized experimental approaches:

• Calcium manipulation strategies:

  • Compare results in calcium-chelated (EGTA/BAPTA) vs. calcium-supplemented conditions

  • Use calcium ionophores (A23187, ionomycin) to elevate intracellular calcium

  • Consider thapsigargin treatment to release ER calcium stores

• Membrane association assays:

  • Subcellular fractionation to track CPNE2 translocation upon calcium flux

  • Liposome binding assays with varying calcium concentrations

  • Live-cell imaging using GFP-tagged CPNE2 during calcium oscillations

• C2 domain functional analysis:

  • Compare wild-type CPNE2 with C2 domain mutants

  • Use calcium-binding site mutants as negative controls

  • Employ domain-swap experiments with other C2 domain proteins

• Phospholipid binding specificity:

  • PIP strip assays with varying calcium concentrations

  • Surface plasmon resonance (SPR) with controlled calcium levels

  • Liposome sedimentation assays with different phospholipid compositions

• Antibody considerations:

  • Ensure antibody recognition is not calcium-dependent

  • Use fixation methods that preserve calcium-dependent interactions

  • Consider epitope accessibility in different conformational states

Understanding CPNE2's calcium-dependent membrane association is critical, as it suggests functional roles in signaling pathways and membrane trafficking events that may be relevant to both normal physiology and disease states .

What are key considerations when using CPNE2 antibodies in disease-related research?

When investigating CPNE2 in disease contexts, researchers should consider:

• Disease-specific expression patterns:

  • CPNE2 has potential implications in diseases related to disruptions in cellular homeostasis:

    • Cancer progression mechanisms

    • Neurological disorders

  • Validate antibody specificity in disease-relevant tissues

• Comparative analysis methodology:

  • Design paired analyses of normal vs. diseased tissues

  • Consider using tissue microarrays for high-throughput screening

  • Implement quantitative image analysis for objective assessment

• Technical considerations for disease tissues:

  • Disease tissues may require modified protocols:

    • Adjusted antigen retrieval for fibrotic tissues

    • Modified fixation protocols for highly necrotic samples

    • Special considerations for calcified tissues

• Controls and validation in disease models:

  • Include gradient of disease progression when available

  • Validate findings across multiple patient samples

  • Correlate protein expression with genetic or transcriptomic data

  • Consider using multiple antibodies to confirm findings

• Application-specific recommendations:

  • For IHC in brain tissue:

    • PACO38718: 1:100 dilution has been validated

    • 17664-1-AP: 1:50-1:500 range with optimization needed

  • For Western blot of disease samples:

    • 17664-1-AP: 1:500-1:1000 dilution recommended

Given CPNE2's involvement in calcium signaling and membrane trafficking, its dysregulation may contribute to disease mechanisms through altered cellular communication or protein localization .

What emerging methodologies might enhance CPNE2 antibody applications?

Several cutting-edge approaches show promise for advancing CPNE2 research:

• Advanced validation methods:

  • CRISPR/Cas9 knockout validation has emerged as a gold standard for antibody specificity testing

  • Multiplexed epitope competition assays for polyclonal antibody characterization

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Single-cell applications:

  • Adapting CPNE2 antibodies for mass cytometry (CyTOF)

  • Single-cell Western blot technologies

  • Imaging mass cytometry for tissue section analysis

• Live-cell dynamics:

  • Nanobody development for live-cell CPNE2 tracking

  • Integrating calcium imaging with CPNE2 visualization

  • FRET-based approaches for studying CPNE2 interactions

• Spatial context preservation:

  • Highly multiplexed imaging using cyclic immunofluorescence

  • Spatial transcriptomics correlated with CPNE2 protein localization

  • 3D tissue clearing techniques combined with CPNE2 immunostaining

These emerging technologies will help address current limitations in studying CPNE2 dynamics and interactions, particularly in the context of calcium signaling and membrane trafficking processes that are central to CPNE2 function .

How should researchers approach contradictory findings when using different CPNE2 antibodies?

When faced with discrepant results using different CPNE2 antibodies:

• Systematic comparative analysis:

  • Test multiple antibodies side-by-side under identical conditions

  • Compare results across different applications (WB, IHC, IF)

  • Document epitope differences between antibodies

• Validation hierarchy implementation:

  • Prioritize results from antibodies validated with knockout controls

  • Consider antibody class differences (monoclonal vs. polyclonal)

  • Evaluate evidence quality for each antibody's validation

• Technical resolution approaches:

  • Optimize conditions for each antibody independently

  • Consider tissue/sample-specific factors affecting epitope accessibility

  • Implement orthogonal techniques to confirm findings

• Literature evaluation strategy:

  • Critically assess antibody validation methods in published studies

  • Note that antibodies failing to recognize their targets have been used in highly cited papers

  • Consider reproducibility issues that may stem from antibody variability