The search results contain no references to "CTPA3 Antibody." Instead, anti-CCP3 antibodies are discussed in the context of predicting inflammatory arthritis (IA) and rheumatoid arthritis (RA) in individuals with musculoskeletal (MSK) symptoms and negative second-generation anti-CCP2 tests . This suggests a possible typographical error or confusion between "CTPA3" and "CCP3."
Anti-CCP3 antibodies are autoantibodies targeting citrullinated peptides, particularly cyclic citrullinated peptides (CCPs). They are used as biomarkers to predict IA progression in at-risk populations.
Studies demonstrate that anti-CCP3 testing improves predictive accuracy for IA in anti-CCP2-negative populations:
Progression Rates:
Outcome | Odds Ratio (OR) at Manufacturer Cutoff | Optimized Cutoff (≥5 units) |
---|---|---|
Self-reported IA | 7.5 (2.3–24.0) | Higher OR observed |
IA Progression | 3.5 (1.2–11.0) | Significantly increased OR |
RA Progression | 2.4 (0.5–18.6) | Associated with RA diagnosis |
Antibody Structure: Anti-CCP3 antibodies share the canonical Y-shaped structure with two heavy and two light chains, featuring hypervariable complementarity-determining regions (CDRs) for antigen binding .
Glycosylation: The Fc region may influence effector functions, though specific data on anti-CCP3 glycosylation patterns are lacking .
Diagnostic Challenges: Low sensitivity at standard thresholds limits clinical utility, prompting exploration of optimized cutoffs (e.g., ≥5 units) .
Research Gaps:
Limited longitudinal data on long-term outcomes.
Mechanistic studies on anti-CCP3-mediated immune modulation are needed.
Antibody Type | Target | Clinical Use Case | Performance (AUC) |
---|---|---|---|
Anti-CCP3 | Citrullinated peptides | IA prediction in anti-CCP2− patients | 0.832–0.887 |
Anti-CCP2 | Citrullinated peptides | RA diagnosis | High specificity |
CTNNA3 | Catenin alpha-3 | Research (muscle cell adhesion) | N/A |
CTPA3 (Carboxy-Terminal Protease A3) belongs to the family of carboxy-terminal proteases that play essential roles in cellular processes. Based on homologous proteins like CtpA in bacterial systems, these proteases are involved in regulating cell separation, envelope integrity, and morphology . In Arabidopsis thaliana, CTPA3 likely participates in protein processing pathways similar to bacterial counterparts.
Studies with CtpA in other organisms demonstrate that it associates with the inner membrane despite lacking obvious signal sequences or transmembrane domains . This suggests CTPA3 may have similar localization patterns in plant systems. Importantly, research indicates these proteases may function as phosphatases that dephosphorylate proteins involved in cell envelope maintenance and division processes.
The CTPA3 antibody (e.g., CSB-PA553428XA01DOA) is typically a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana CTPA3 protein . Its technical specifications include:
Production Host: Rabbit
Immunogen: Recombinant Arabidopsis thaliana CTPA3 protein
Species Reactivity: Arabidopsis thaliana
Applications: ELISA, Western Blot
Form: Liquid
Purification Method: Antigen Affinity Purified
Storage Buffer: 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative
The antibody is designed for research applications in plant molecular biology and is typically characterized through validation in Western blot and ELISA applications.
For optimal Western blotting with CTPA3 antibody, researchers should consider that protein conformation may be critical for epitope recognition, similar to other antibodies in this family . A recommended protocol includes:
Sample preparation in non-reducing buffer to preserve epitope structure
SDS-PAGE separation (typically 10-12% gels)
Transfer to PVDF or nitrocellulose membrane
Blocking with 5% non-fat milk or BSA in TBS-T for 1 hour at room temperature
Primary antibody incubation (1:500-1:1000 dilution) overnight at 4°C
Secondary antibody (anti-rabbit HRP) incubation for 1 hour at room temperature
Four 5-minute washes with TBS-0.5% Tween 20
Chemiluminescent detection
It's advisable to include positive controls (recombinant CTPA3) and negative controls (lysates from CTPA3 knockout lines) to validate specificity.
Based on protocols for similar antibodies, immunoprecipitation with CTPA3 antibody requires careful optimization:
Prepare fresh plant tissue lysates in a non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, plus protease inhibitors)
Pre-clear lysate with Protein A/G beads for 1 hour at 4°C
Incubate cleared lysate with CTPA3 antibody (2-5 μg per 500 μg protein) overnight at 4°C
Add Protein A beads and incubate for 2-4 hours at 4°C
Wash beads extensively (4-5 times) with lysis buffer
Elute bound proteins by boiling in sample buffer
Analyze by Western blotting with a detection antibody
For complex tissues or weak interactions, crosslinking with DSP (dithiobis[succinimidyl propionate]) prior to lysis can help preserve protein interactions. Similar to techniques used with other antibodies, proper validation involves confirming pull-down specificity through immunoblotting of precipitated material .
Comprehensive validation of CTPA3 antibody specificity requires multiple approaches:
Genetic validation: Compare signal between wild-type and CTPA3 knockout/knockdown plant lines
Peptide competition: Pre-incubate antibody with immunizing peptide/protein to demonstrate signal ablation
Molecular weight verification: Confirm detection at expected molecular weight (corresponding to CTPA3)
Cross-reactivity assessment: Test antibody against closely related CTP family members
Immunoprecipitation-mass spectrometry: Confirm that immunoprecipitated proteins include CTPA3
Heterologous expression: Test against recombinant CTPA3 expressed in bacterial or insect cell systems
These validation steps are critical for ensuring experimental reproducibility and avoiding misinterpretation of results, particularly when studying proteins with sequence similarity to CTPA3.
For immunolocalization of CTPA3 in plant tissues, several factors must be considered:
Fixation method: Use 4% paraformaldehyde to preserve both protein epitopes and cellular architecture
Antigen retrieval: Optimize based on fixation method (citrate buffer at pH 6.0 often works well)
Permeabilization: Use 0.1-0.5% Triton X-100 to allow antibody access while preserving cellular structures
Background reduction: Pre-absorb antibody with plant extract from CTPA3 knockout tissue
Co-localization markers: Include organelle-specific markers to determine precise subcellular localization
Controls: Include secondary-only and peptide competition controls
Given that bacterial CtpA has been shown to localize to the division site during specific cell cycle stages , particular attention should be paid to potential cell cycle-dependent localization patterns of CTPA3 in plant cells.
When multiple bands appear in Western blots with CTPA3 antibody, systematic analysis is required:
Expected bands: The primary CTPA3 band should match the predicted molecular weight
Post-translational modifications: Higher molecular weight bands may represent phosphorylated or otherwise modified forms
Proteolytic fragments: Lower molecular weight bands may indicate protein degradation or processing
Cross-reactivity: Some bands may represent related CTP family proteins
Verification approach: Compare band patterns between tissues/conditions and use immunoprecipitation followed by mass spectrometry to identify proteins in each band
A systematic approach to band identification, including treatment with phosphatases or proteases, can help distinguish between specific signal and cross-reactivity.
When experiments with CTPA3 antibody fail to yield expected results, consider these troubleshooting approaches:
Antibody activity: Test freshly prepared antibody dilutions and verify storage conditions haven't compromised activity
Epitope accessibility: If protein conformation affects recognition, adjust lysis and sample preparation conditions
Protocol optimization: Systematically vary antibody concentration, incubation time/temperature, and wash stringency
Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers) to reduce background
Sample quality: Ensure protein integrity by using fresh samples with appropriate protease inhibitors
Expression levels: Verify CTPA3 expression in your experimental system, as low abundance may require enrichment
Detection method: Switch between chemiluminescence, fluorescence, or chromogenic detection based on expected abundance
A systematic approach to troubleshooting involves changing one variable at a time and documenting outcomes for each modification.
CTPA3 antibody can be leveraged to investigate potential roles in plant stress responses through:
Protein expression analysis: Quantify CTPA3 levels across different stress conditions (drought, salt, pathogens) using quantitative Western blotting
Phosphorylation status: Use phosphatase treatments combined with CTPA3 immunoblotting to assess stress-induced post-translational modifications
Protein-protein interactions: Perform co-immunoprecipitation under stress conditions to identify stress-specific interaction partners
Subcellular relocalization: Track CTPA3 localization changes during stress responses using immunofluorescence
Chromatin association: If CTPA3 has nuclear functions, chromatin immunoprecipitation can reveal DNA-binding properties
Turnover rate analysis: Cycloheximide chase experiments with CTPA3 antibody detection can reveal changes in protein stability during stress
These approaches can help determine whether CTPA3 functions change during stress adaptation in plants, similar to how bacterial CtpA affects cell envelope integrity under stress conditions .
Integrating antibody-based detection with genetic manipulation provides powerful insights:
CRISPR-engineered lines: Compare CTPA3 antibody labeling in wild-type versus CRISPR-modified plants with specific domain deletions or mutations
Complementation studies: Use the antibody to verify expression levels in transgenic complementation lines
Inducible systems: Track protein accumulation in inducible expression systems for kinetic studies
Structure-function analysis: Correlate antibody-detected protein levels with phenotypic outcomes in plants expressing mutated versions
Tissue-specific expression: Combine immunohistochemistry with tissue-specific promoters to correlate localized expression with function
Protein complex analysis: Use antibody to verify complex formation in lines expressing tagged potential interactors
One particularly valuable approach involves creating active site mutants similar to those used in bacterial studies and using the antibody to confirm expression while correlating with phenotypic outcomes.
Research on CtpA homologs suggests potential roles in immunity, where antibody-based approaches can reveal:
Pathogen-induced changes: Monitor CTPA3 abundance, modification, and localization during pathogen challenge
Protein processing events: Track potential proteolytic events during immune responses
Immune complex formation: Identify associations with known immune receptors via co-immunoprecipitation
Guard hypothesis testing: Determine if CTPA3 is monitored by immune receptors as a potential pathogen target
DAMPs signaling: Investigate if CTPA3 fragments serve as damage-associated molecular patterns
Studies with bacterial pathogens lacking functional ctpA show attenuated virulence and altered interactions with host immunity , suggesting plant CTPA3 may similarly interface with immune pathways.
When extending CTPA3 antibody use beyond Arabidopsis:
Sequence homology analysis: Determine conservation of the antibody epitope in target species
Validation requirements: Perform Western blot validation in each new species before proceeding to complex applications
Extraction buffer optimization: Adjust lysis conditions for species-specific differences in cell wall composition
Fixation protocol adjustments: Modify fixation times and conditions for different tissue types
Species-specific controls: Include appropriate positive and negative controls from the target species
Cross-reactivity assessment: Test for unexpected cross-reactivity with species-specific proteins
Since the CTPA3 antibody was raised against Arabidopsis thaliana protein , its performance will likely correlate with epitope conservation. Preliminary sequence alignment analysis and Western blot validation should precede detailed studies in non-Arabidopsis species.
Advanced protein interaction studies with CTPA3 antibody include:
Proximity labeling: Couple CTPA3 antibody with BioID or APEX2 systems to identify neighboring proteins in vivo
Super-resolution microscopy: Use fluorophore-conjugated CTPA3 antibody for nanoscale colocalization studies
Interactome mapping: Perform sequential immunoprecipitations to identify higher-order protein complexes
Dynamic interactome analysis: Track interaction changes across developmental stages or stress conditions
In situ proximity ligation: Visualize protein-protein interactions directly in fixed plant tissues
Cross-linking mass spectrometry: Identify interaction interfaces by cross-linking followed by immunoprecipitation
Bacterial studies show that CtpA interacts with proteins involved in cell envelope maintenance and division , suggesting CTPA3 likely participates in multiprotein complexes that could be characterized using these approaches.
Emerging technologies and approaches that could enhance CTPA3 antibody applications include:
Nanobody development: Generating small single-domain antibodies against CTPA3 for improved tissue penetration
Intrabody applications: Expressing antibody fragments in vivo to modulate CTPA3 function
BiFC-compatible fragments: Developing split fluorescent protein-tagged antibody fragments for interaction visualization
Single-cell proteomics: Adapting CTPA3 antibody for mass cytometry or microfluidic antibody capture
Quantitative interactomics: Implementing stable isotope labeling for quantitative analysis of CTPA3 interactions
Structural studies: Using antibody fragments to facilitate crystallization of CTPA3 for structure determination
Synthetic circuit engineering: Incorporating CTPA3 and its antibody into synthetic biology applications in plants
The development of these advanced applications could significantly expand our understanding of CTPA3 function in plant development, stress responses, and immunity.