COPT5 Antibody

What is COPT5 and why is it important to develop antibodies against it?

COPT5 is a copper transporter protein that plays a critical role in copper homeostasis and accumulation in plant tissues, particularly in shoots. Research has demonstrated that COPT5, along with other copper transporters like HMA5 and COPT1, is induced during viral infections such as Rice Stripe Virus (RSV) to promote copper accumulation . This copper accumulation strengthens antiviral defenses through specific molecular pathways. Developing antibodies against COPT5 is essential for investigating its expression patterns, subcellular localization, and protein-protein interactions, which collectively advance our understanding of copper-mediated defense mechanisms in plants.

How do COPT5 antibodies differ from other copper transporter antibodies?

COPT5 antibodies are specifically designed to recognize unique epitopes in the COPT5 protein structure that distinguish it from other copper transporter family members. While the COPT family contains seven members in rice (COPT1-COPT7) with varying expression patterns during viral infection, COPT5 shows distinct upregulation during RSV infection compared to other family members like COPT7, which is downregulated . Antibodies targeting COPT5 must be validated for specificity by confirming they do not cross-react with other COPT proteins, particularly COPT1, which shares functional similarities. This specificity validation typically involves Western blot analysis using recombinant proteins or tissues from knockout lines to ensure accurate research outcomes.

What experimental techniques benefit most from COPT5 antibody applications?

COPT5 antibodies enable multiple experimental approaches crucial for understanding copper transport mechanisms:

• Immunolocalization studies: Using immunohistochemistry and immunofluorescence techniques to determine the tissue and subcellular distribution of COPT5, providing insights into its functional domains within plant cells.

• Protein quantification: Western blotting for measuring COPT5 protein levels during different developmental stages or stress conditions, particularly during viral infections when expression is significantly altered .

• Protein-protein interaction studies: Co-immunoprecipitation experiments to identify proteins that interact with COPT5, potentially revealing regulatory partners in copper homeostasis pathways.

• Chromatin immunoprecipitation (ChIP): For researchers investigating potential transcription factors that regulate COPT5 expression during stress responses.

These techniques collectively provide comprehensive data on COPT5 regulation, expression, and function in plant physiology and pathogen responses.

How can researchers address epitope masking issues when detecting COPT5 in complex protein samples?

Epitope masking represents a significant challenge when detecting membrane-bound proteins like COPT5, particularly due to their association with lipid-rich environments and potential protein-protein interactions. To overcome this limitation:

• Sample preparation optimization: Researchers should test multiple protein extraction buffers containing different detergents (CHAPS, Triton X-100, or n-dodecyl β-D-maltoside) at varying concentrations to efficiently solubilize membrane compartments while preserving epitope structures.

• Antigenic retrieval methods: When performing immunohistochemistry on fixed tissues, heat-induced epitope retrieval using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) can improve antibody access to masked epitopes.

• Multiple antibody approach: Utilizing antibodies targeting different regions of COPT5 (N-terminal, C-terminal, and internal domains) provides complementary detection methods to overcome conformational masking issues.

• Native versus denatured detection: Comparing results from native PAGE and SDS-PAGE Western blotting can reveal whether epitope accessibility differs under native versus denaturing conditions, informing optimal detection protocols.

This systematic approach ensures reliable COPT5 detection even in complex biological samples where protein conformation or interactions might obscure antibody binding sites.

What methodological approaches can resolve contradictory data on COPT5 function in different experimental systems?

When researchers encounter contradictory results regarding COPT5 function across different experimental systems, several methodological strategies can help resolve these discrepancies:

• Genetic complementation studies: Reintroducing wild-type or mutated COPT5 into knockout lines can confirm whether observed phenotypes are directly attributable to COPT5 function rather than secondary effects or compensatory mechanisms.

• Cross-species validation: Examining COPT5 function across multiple plant species can identify conserved versus species-specific functions, explaining apparently contradictory results from different model systems.

• Tissue-specific and developmental analysis: Since COPT5 may function differently across tissues and developmental stages, conducting temporally and spatially resolved expression studies using COPT5 antibodies can reconcile seemingly conflicting data.

• Integration of in vitro and in vivo approaches: Combining biochemical assays of copper transport activity with whole-organism phenotyping provides complementary evidence that can explain contradictions arising from examining COPT5 in isolated contexts.

• Rigorous controls for antibody specificity: Using tissues from COPT5 knockout mutants as negative controls in immunodetection experiments ensures that observed signals are specific and not arising from antibody cross-reactivity with related proteins .

These approaches collectively provide a framework for resolving contradictions and establishing consensus on COPT5 function across experimental systems.

How does COPT5's role in antiviral defense intersect with its copper transport function?

The intersection between COPT5's copper transport function and its role in antiviral defense represents a sophisticated regulatory network that researchers can investigate using COPT5 antibodies. This relationship is characterized by several key mechanisms:

• Copper-dependent regulation of defense pathways: COPT5-mediated copper accumulation in shoots during viral infection suppresses the transcriptional activation of microRNA miR528 by inhibiting SPL9 protein levels . COPT5 antibodies can be used in ChIP experiments to identify if transcription factors directly involved in defense responses interact with the COPT5 promoter upon viral infection.

• Copper transport and ROS signaling: COPT5 contributes to antiviral defense by affecting downstream copper-binding proteins like AO (L-ascorbate oxidase), which regulates reactive oxygen species (ROS) accumulation . Researchers can use COPT5 antibodies in conjunction with activity assays to correlate COPT5 protein levels with AO activity and ROS production during infection.

• Protein-protein interaction network: Co-immunoprecipitation using COPT5 antibodies followed by mass spectrometry analysis can reveal whether COPT5 physically interacts with components of known antiviral signaling pathways, providing mechanistic insights into how copper transport interfaces with defense signaling.

• Structure-function relationship: Utilizing site-directed mutagenesis to alter copper-binding domains in COPT5, followed by immunodetection with COPT5 antibodies, can determine whether copper binding capacity is directly linked to COPT5's ability to activate defense responses.

This multifaceted approach enabled by COPT5 antibodies allows researchers to dissect the complex relationship between copper homeostasis and viral resistance mechanisms.

What are the optimal validation steps to ensure COPT5 antibody specificity?

Rigorous validation of COPT5 antibodies is critical for research integrity. The following comprehensive validation protocol ensures antibody specificity:

• Genetic controls: Test antibodies on samples from wild-type plants and COPT5 knockout mutants (copt5) to confirm signal absence in mutants . This represents the gold standard for validation.

• Peptide competition assays: Pre-incubate the antibody with excess synthetic peptide used as the immunogen before application to samples. Specific antibodies will show significantly reduced signal when blocked with the immunizing peptide.

• Cross-reactivity assessment: Test the antibody against recombinant proteins of all COPT family members (COPT1-COPT7) to ensure it does not recognize related transporters, particularly COPT1 which shares functional similarities with COPT5 .

• Multiple antibody comparison: Validate results using antibodies raised against different epitopes of COPT5 (similar to approaches used for COPS5 antibodies ), which should yield consistent patterns if specific.

• Signal correlation with mRNA expression: Compare protein detection patterns with RT-qPCR data for COPT5 transcripts across tissues and conditions, particularly during viral infection when expression changes are well-documented .

• Immunoprecipitation followed by mass spectrometry: Confirm that immunoprecipitated protein is indeed COPT5 through peptide identification using mass spectrometry.

These validation steps collectively establish the reliability and specificity of COPT5 antibodies for downstream applications.

What are the recommended protocols for optimizing Western blot detection of COPT5?

Optimizing Western blot detection of COPT5 requires careful consideration of its membrane protein nature and expression levels. The following protocol modifications enhance detection sensitivity and specificity:

Sample Preparation:

• Use fresh tissue samples when possible, extracting proteins in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• For membrane fraction enrichment, perform ultracentrifugation (100,000 × g for 1 hour) after initial tissue homogenization and resuspend pellets in buffer containing 0.5% n-dodecyl β-D-maltoside.

Gel Electrophoresis Conditions:

• Use gradient gels (4-15%) to better resolve membrane proteins.

• Load positive controls including recombinant COPT5 protein alongside experimental samples.

• Include samples from copt5 mutant plants as negative controls .

Transfer and Detection:

• Perform wet transfer at 30V overnight at 4°C to ensure complete transfer of membrane proteins.

• Block membranes with 5% non-fat dry milk in TBST containing 0.05% Tween-20.

• Incubate with primary COPT5 antibody (1:1000 dilution) overnight at 4°C.

• Wash membranes extensively (5 × 5 minutes) with TBST before secondary antibody incubation.

• Use enhanced chemiluminescence detection with extended exposure times (up to 10 minutes) for low-abundance samples.

Troubleshooting Common Issues:

• For weak signals, reduce washing stringency and increase antibody concentration to 1:500.

• For high background, increase blocking time to 2 hours and add 0.1% BSA to antibody dilution buffer.

• For multiple bands, validate using peptide competition assays to identify specific COPT5 bands.

This optimized protocol significantly improves detection reliability for COPT5 in plant tissue samples.

How should researchers approach co-immunoprecipitation experiments using COPT5 antibodies?

Co-immunoprecipitation (Co-IP) experiments using COPT5 antibodies require specialized approaches due to the membrane-bound nature of COPT5 and the complexity of copper transport complexes. The following protocol adaptations maximize success:

Pre-Immunoprecipitation Considerations:

• Crosslinking option: Consider using membrane-permeable crosslinkers like DSP (dithiobis[succinimidylpropionate]) at 1-2 mM for 30 minutes prior to cell lysis to stabilize transient protein interactions.

• Detergent selection: Test multiple detergents for solubilization, with 1% digitonin or 0.5% n-dodecyl β-D-maltoside often preserving protein-protein interactions better than stronger detergents like SDS.

• Buffer composition: Include 5-10 μM copper sulfate in extraction buffers to stabilize copper-dependent protein interactions.

Immunoprecipitation Protocol:

• Prepare protein extracts in IP buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, and protease inhibitor cocktail.

• Pre-clear lysates with protein A/G beads for 1 hour at 4°C.

• Incubate pre-cleared lysates with COPT5 antibody (5 μg per 1 mg of total protein) overnight at 4°C with gentle rotation.

• Add protein A/G beads and incubate for 3 hours at 4°C.

• Wash beads 5 times with washing buffer (same as IP buffer but with 0.1% NP-40).

• Elute bound proteins by boiling in 2× SDS sample buffer or using gentle elution with antibody-specific peptides.

Validation and Controls:

• Include non-immune IgG as a negative control processed identically to experimental samples.

• Use lysates from copt5 knockout plants as specificity controls .

• Perform reciprocal Co-IPs when possible, immunoprecipitating with antibodies against suspected interaction partners and blotting for COPT5.

• Confirm that interactions are consistent with subcellular localization data from immunofluorescence studies.

Following this specialized protocol increases the likelihood of identifying genuine COPT5 interaction partners while minimizing artifacts.

How do antibody-based approaches for studying COPT5 compare with genetic approaches?

Antibody-based and genetic approaches offer complementary insights into COPT5 function, with distinct advantages and limitations summarized in the following comparative analysis:

Research on COPT5's role in antiviral defense demonstrates this complementarity, where genetic approaches identified copt5 mutants with compromised viral resistance, while antibody-based approaches could determine if this correlates with altered protein localization or interaction networks . The most robust experimental designs incorporate both methodologies to provide multiple lines of evidence regarding COPT5 function.

What are the considerations for studying COPT5 protein-protein interactions in different experimental systems?

Investigating COPT5 protein-protein interactions presents unique challenges across experimental systems, requiring tailored approaches:

In Planta Systems:

• Native expression contexts: Use COPT5 antibodies for co-immunoprecipitation from plant tissues under relevant conditions (e.g., viral infection) to capture physiologically relevant interactions .

• Membrane microdomains: Consider using membrane fractionation prior to immunoprecipitation, as COPT5 may reside in specific membrane domains that influence its interaction landscape.

• Validation approach: Confirm interactions through multiple methods, including bimolecular fluorescence complementation (BiFC) and co-localization studies using COPT5 antibodies for immunofluorescence.

• Stimulus-dependent interactions: Examine how viral infection or copper status affects COPT5 interactions, particularly with the SPL9-miR528-AO pathway components .

Heterologous Expression Systems:

• Expression optimization: When expressing COPT5 in yeast or mammalian cells, codon optimization may be necessary, and expression levels should be validated using COPT5 antibodies.

• Membrane protein considerations: Include appropriate tags for affinity purification that don't interfere with membrane insertion or protein folding.

• Cross-species interactions: Assess whether interactions identified in heterologous systems also occur in planta using COPT5 antibodies in native contexts.

• Copper supplementation: Include physiologically relevant copper concentrations in media to promote native conformation and interactions.

In Vitro Reconstitution:

• Protein purification: For pull-down assays, purify full-length COPT5 in detergent micelles or nanodiscs to maintain native conformation.

• Direct vs. indirect interactions: Use purified components to distinguish direct binding partners from complex components.

• Binding kinetics: Employ surface plasmon resonance or microscale thermophoresis with purified components to quantify interaction affinities and kinetics.

By tailoring approaches to each experimental system and validating across multiple platforms, researchers can build a comprehensive and reliable interaction network for COPT5.

How can researchers accurately quantify COPT5 protein levels during viral infection studies?

Accurate quantification of COPT5 protein during viral infection studies requires specialized approaches to address the challenges of membrane protein analysis and dynamic expression changes:

Sample Preparation Considerations:

• Timing of collection: Establish a detailed time course following viral inoculation (e.g., RSV) with sampling at 0, 12, 24, 48, 72, and 96 hours post-infection to capture the full expression dynamics .

• Tissue separation: Separately analyze shoots and roots, as COPT5 shows tissue-specific accumulation patterns during viral response .

• Subcellular fractionation: Prepare membrane-enriched fractions to concentrate COPT5 protein for more sensitive detection.

• Protein extraction buffer: Use buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail with 5 mM EDTA to inhibit metalloproteases.

Quantification Methods:

• Western blot with internal standards: Include recombinant COPT5 protein standards at known concentrations (e.g., 0.1, 0.5, 1, 5, and 10 ng) on each gel to generate standard curves for absolute quantification.

• Normalization strategy:

  • For total protein normalization, use stain-free gel technology or REVERT total protein stain rather than single housekeeping proteins.

  • For membrane protein-specific normalization, probe for stable membrane proteins like H⁺-ATPase that aren't altered by viral infection.

• ELISA development: Consider developing a sandwich ELISA using two antibodies targeting different COPT5 epitopes for high-throughput quantification across multiple samples.

• Mass spectrometry-based quantification: For absolute quantification, implement parallel reaction monitoring (PRM) mass spectrometry using isotopically labeled peptide standards corresponding to unique COPT5 sequences.

Data Analysis Approach:

• Use at least three biological replicates with technical duplicates for statistical robustness.

• Perform two-way ANOVA to assess the interaction between viral infection and time course factors.

• Present data as fold-change relative to uninfected controls at each time point.

• Correlate protein levels with corresponding mRNA expression (measured by RT-qPCR) to identify potential post-transcriptional regulation mechanisms.

This comprehensive approach ensures accurate quantification of COPT5 protein dynamics during viral infection, enabling correlation with phenotypic outcomes and mechanistic insights.

How might COPT5 antibodies contribute to understanding cross-talk between copper transport and other metal homeostasis pathways?

COPT5 antibodies offer powerful tools for investigating the complex interplay between copper transport and other metal homeostasis pathways, opening several innovative research directions:

• Co-localization studies: Using COPT5 antibodies in combination with antibodies against iron, zinc, or manganese transporters in dual immunofluorescence experiments can reveal potential co-localization or mutual exclusion in membrane microdomains, suggesting functional relationships.

• Metal-dependent protein complex analysis: Implementing co-immunoprecipitation with COPT5 antibodies under varying metal conditions (copper deficiency, excess, or combined metal stresses) followed by proteomic analysis can identify metal-specific protein interaction networks.

• Post-translational modification profiling: Immunoprecipitating COPT5 using specific antibodies followed by mass spectrometry analysis can reveal how phosphorylation, ubiquitination, or other modifications change under different metal stress conditions, potentially identifying regulatory cross-talk mechanisms.

• Conformational antibodies: Developing conformation-specific antibodies that recognize COPT5 only when bound to copper could provide tools to monitor metal occupancy in vivo, similar to approaches used for other transporters.

• Tissue-specific metal homeostasis: Combining immunohistochemistry using COPT5 antibodies with synchrotron X-ray fluorescence microscopy for metal localization can correlate COPT5 expression patterns with copper distribution across tissues during development or stress responses.

This integrative approach using COPT5 antibodies will help decipher how plants coordinate multiple metal homeostasis pathways during development and in response to environmental challenges, potentially revealing novel intervention points for improving crop resilience.

What potential exists for developing COPT5 antibodies that distinguish between different functional states?

The development of state-specific COPT5 antibodies represents an emerging frontier with significant research potential. These specialized antibodies could distinguish between:

• Metal-bound versus apo states: Generating antibodies against conformational epitopes that are exposed only when COPT5 is bound to copper or in its metal-free state. This approach would require:

  • Immunizing with purified COPT5 protein stabilized in specific conformational states

  • Screening antibody clones for differential recognition of copper-bound versus copper-free COPT5

  • Validating specificity using COPT5 mutants with altered copper binding capacity

• Active versus inactive transport conformations: Developing antibodies that specifically recognize COPT5 in its inward-facing or outward-facing conformations, similar to approaches used for other membrane transporters:

  • Designing peptide immunogens mimicking regions that undergo conformational changes during transport

  • Using structural information to identify exposed epitopes unique to each conformational state

  • Employing conformation-trapping agents during immunization and screening

• Phosphorylation-dependent states: Creating antibodies that specifically recognize phosphorylated COPT5 at key regulatory residues:

  • Performing bioinformatic analysis to identify potential regulatory phosphorylation sites

  • Synthesizing phosphopeptides corresponding to these regions for immunization

  • Testing phospho-specific antibodies in contexts where COPT5 regulation is likely, such as during viral infection

• Oligomerization-specific antibodies: Developing antibodies that specifically recognize COPT5 monomers versus oligomers:

  • Using chemical cross-linking to stabilize different oligomeric states for immunization

  • Screening for antibodies that selectively bind interfaces formed during oligomerization

  • Validating with size exclusion chromatography and native PAGE analysis

These state-specific antibodies would revolutionize our understanding of COPT5 regulation by allowing researchers to track the functional status of the transporter in real-time during stress responses, particularly during antiviral defense activation .

How can COPT5 antibodies contribute to translational research bridging plant and human copper transport disorders?

COPT5 antibodies have significant potential to advance translational research connecting plant and human copper transport mechanisms, particularly given the evolutionary conservation of copper homeostasis pathways:

• Comparative structure-function analysis: Using COPT5 antibodies to immunoprecipitate plant COPT5 for structural studies can provide insights applicable to human copper transporters like CTR1 and CTR2, which share functional similarities:

  • Purifying COPT5-antibody complexes for cryo-EM structural analysis

  • Comparing structural features with human CTR proteins to identify conserved functional domains

  • Testing whether human copper transporter antibodies show cross-reactivity with plant COPT proteins to identify highly conserved epitopes

• Model system development: COPT5 antibodies enable the validation of plant models for studying copper-related human diseases:

  • Creating transgenic plants expressing human copper transporters and using both COPT5 and human-specific antibodies to study their localization and function

  • Using immunofluorescence to determine if human copper transporters correctly localize in plant cells

  • Examining if plant antiviral mechanisms involving copper have parallels in human antiviral responses

• Drug discovery applications: Plant systems with well-characterized COPT proteins (detected using specific antibodies) can serve as preliminary screening platforms for compounds affecting copper transport:

  • Screening chemical libraries for compounds that alter COPT5 protein levels or localization as detected by antibodies

  • Testing promising compounds in mammalian cell models of copper transport disorders

  • Developing high-throughput immunoassays using COPT5 antibodies for large-scale compound screening

• Biomarker development: Insights from plant COPT5 regulation during stress could inform biomarker development for human copper-related disorders:

  • Identifying stress-induced post-translational modifications of COPT5 using specific antibodies

  • Investigating if analogous modifications occur in human copper transporters under disease conditions

  • Developing diagnostic antibodies targeting specific modified forms of human copper transporters

This translational approach leverages the accessibility and genetic tractability of plant systems, with COPT5 antibodies serving as essential tools to bridge fundamental discoveries in plant copper transport to applications in human health.