CCAR2 Monoclonal Antibody

What is CCAR2 and what is its significance in cellular function?

CCAR2 (Cell Cycle and Apoptosis Regulator 2) is a multifunctional protein primarily localized in the nucleus and cytoplasm with a significant role in cell cycle regulation and apoptosis . The canonical human CCAR2 protein consists of 923 amino acid residues with a molecular mass of approximately 102.9 kDa, although the observed molecular weight in experimental conditions often appears around 130 kDa . CCAR2 functions as a core component of the DBIRD complex, which operates at the interface between mRNP particles and RNA polymerase II (RNAPII) . This complex is critical for integrating transcript elongation with alternative splicing regulation, specifically affecting local transcript elongation rates and alternative splicing of exons in (A+T)-rich DNA regions . CCAR2 expression has been observed to increase progressively in gastric carcinoma tissue as the disease advances, suggesting its potential involvement in cancer progression mechanisms .

What are the common synonyms and alternative designations for CCAR2?

Researchers should be aware of several alternative designations for CCAR2 when reviewing literature or searching databases. The most common synonyms include "deleted in breast cancer 1" (DBC1), "cell division cycle and apoptosis regulator protein 2," and KIAA1967 . The UniProt primary accession number for human CCAR2 is Q8N163, with the entry name CCAR2_HUMAN . In genomic databases, CCAR2 is identified by GeneID 57805 and may be referenced by NCBI accession number NP_066997.3 . Understanding these alternative designations is essential for comprehensive literature searches and avoiding confusion when comparing research findings across different studies.

What are the typical applications for CCAR2 monoclonal antibodies in research?

CCAR2 monoclonal antibodies have several established applications in molecular and cellular research. The most common applications include:

• Western Blotting (WB): Used to detect and quantify CCAR2 protein expression in cell or tissue lysates .

• Immunohistochemistry (IHC): Applied to visualize CCAR2 distribution in tissue sections, particularly useful for cancer studies .

• Immunofluorescence (IF): Employed to determine subcellular localization and expression patterns of CCAR2 in cultured cells .

• ELISA: Utilized for quantitative detection of CCAR2 in solution .

• Microarray Analysis: Some monoclonal antibodies, such as CPTC-KIAA1967-1, are suitable for protein microarray applications, enabling high-throughput screening .

Over 50 citations in the scientific literature describe the use of CCAR2 antibodies in various research applications, demonstrating their established utility in advancing understanding of this protein's functions .

How do polyclonal and monoclonal CCAR2 antibodies differ in research applications?

When selecting between polyclonal and monoclonal CCAR2 antibodies, researchers should consider several key differences that impact experimental outcomes:

Monoclonal CCAR2 antibodies, such as CPTC-KIAA1967-1 (mouse IgG2b), offer high specificity as they recognize a single epitope on the CCAR2 protein . This specificity provides consistent results across experiments with minimal batch-to-batch variation, making them ideal for applications requiring precise epitope targeting and reproducible results. Monoclonal antibodies are particularly valuable for detecting specific isoforms or post-translational modifications of CCAR2.

The choice between monoclonal and polyclonal antibodies should be guided by the specific research question, required sensitivity and specificity, and the particular application methodology employed.

What factors should be considered when selecting a CCAR2 antibody for specific experimental applications?

When selecting a CCAR2 antibody for research, several critical factors should be evaluated to ensure optimal experimental outcomes:

• Immunogen design: Consider whether the antibody was raised against the full-length protein (amino acids 1-923) or a specific region (e.g., amino acids 654-923) . Full-length immunogens may provide broader epitope recognition, while fragment-specific antibodies might offer more targeted detection of specific domains or isoforms.

• Host species and clonality: Mouse monoclonal or rabbit polyclonal options are available . Selection should account for potential cross-reactivity issues in your experimental system and the intended application.

• Validated applications: Verify that the antibody has been specifically validated for your intended application (WB, IF, IHC, ELISA) . For example, CPTC-KIAA1967-1 is recommended for ELISA, microarray, and Western blot applications but may not be optimal for all IHC protocols .

• Species reactivity: Confirm reactivity with your experimental species. Some antibodies react only with human CCAR2, while others cross-react with mouse and rat homologs .

• Observed molecular weight: Note that while the calculated molecular weight of CCAR2 is approximately 103 kDa, it often appears at around 130 kDa in experimental conditions, potentially due to post-translational modifications . Verify that the antibody detects the appropriate band size in your experimental system.

• Required dilutions: Consider the recommended working dilution for your specific application (e.g., 1/500-1/2000 for Western blot, 1/50-1/200 for IHC-P) , as this impacts antibody consumption and experimental cost.

What are the recommended protocols for using CCAR2 monoclonal antibodies in Western blotting?

For optimal results when using CCAR2 monoclonal antibodies in Western blotting, the following methodological considerations should be addressed:

Sample preparation:

• Prepare cell or tissue lysates using standard protocols with protease inhibitors to prevent degradation of CCAR2 (MW: 103 kDa calculated, typically observed at 130 kDa) .

• Include phosphatase inhibitors if phosphorylated forms of CCAR2 are of interest.

• For subcellular analysis, consider separate nuclear and cytoplasmic fractionation as CCAR2 localizes to both compartments .

Electrophoresis and transfer:

• Use 8-10% SDS-PAGE gels to provide optimal resolution in the 100-130 kDa range.

• Ensure complete transfer of high molecular weight proteins by using low methanol concentration in transfer buffer or considering wet transfer methods.

Antibody incubation:

• For monoclonal antibodies like CPTC-KIAA1967-1, begin with the manufacturer's recommended dilution (typically 1:500 to 1:2000) .

• Incubate primary antibody at 4°C overnight for optimal specific binding.

• Use appropriate secondary antibodies matching the host species (anti-mouse for CPTC-KIAA1967-1 or anti-rabbit for polyclonal alternatives) .

Detection and troubleshooting:

• If the observed band deviates from the expected 130 kDa, consider the possibility of detecting different isoforms (up to 2 have been reported) or post-translational modifications.

• For weak signals, optimize antibody concentration, consider longer exposure times, or implement signal enhancement systems.

• To reduce background, increase blocking time or concentration, or adjust washing stringency.

How should CCAR2 antibodies be used for immunohistochemistry and immunofluorescence applications?

When employing CCAR2 antibodies for immunohistochemistry (IHC) and immunofluorescence (IF) applications, researchers should follow these methodological guidelines for optimal visualization:

Tissue/cell preparation:

• For IHC: Use formalin-fixed, paraffin-embedded (FFPE) tissues with appropriate antigen retrieval techniques to expose CCAR2 epitopes that may be masked during fixation.

• For IF: Fix cultured cells with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100 to allow antibody access to nuclear and cytoplasmic CCAR2.

Antibody dilution and incubation:

• For IHC: Begin with dilutions of 1:50 to 1:200 for polyclonal antibodies . Monoclonal antibodies may require different dilutions based on manufacturer recommendations.

• For IF: Start with similar dilutions but be prepared to optimize based on signal intensity.

• Incubate primary antibodies at 4°C overnight for most applications to enhance specific binding while reducing background.

Controls and validation:

• Always include positive control tissues known to express CCAR2, such as gastric carcinoma tissue where CCAR2 expression has been documented to increase with disease progression .

• Include negative controls (omitting primary antibody) to assess non-specific binding of secondary detection systems.

• Consider using siRNA knockdown controls in cell-based experiments to validate antibody specificity.

Signal detection and analysis:

• For IHC: Both DAB (brown) and AEC (red) chromogens are suitable for visualizing CCAR2.

• For IF: CCAR2 typically shows both nuclear and cytoplasmic localization patterns . Counter-stain nuclei with DAPI to clearly distinguish nuclear from cytoplasmic staining.

• When analyzing results, note that CCAR2 expression patterns may vary with cell cycle stage or in response to cellular stress conditions.

How can CCAR2 antibodies be used to investigate its role in the DBIRD complex and RNA processing?

CCAR2 serves as a core component of the DBIRD complex, which integrates transcript elongation with alternative splicing regulation . To investigate this complex relationship using CCAR2 antibodies, researchers can implement the following advanced methodological approaches:

Chromatin immunoprecipitation (ChIP) assays:

• Use CCAR2 monoclonal antibodies to immunoprecipitate chromatin-bound CCAR2 and associated DNA.

• Map CCAR2 binding sites genome-wide in relation to (A+T)-rich DNA regions where the DBIRD complex affects alternative splicing.

• Combine with RNA polymerase II ChIP to assess co-localization at transcriptionally active regions.

Co-immunoprecipitation (Co-IP) studies:

• Immunoprecipitate CCAR2 using specific monoclonal antibodies to isolate the entire DBIRD complex.

• Analyze co-precipitated proteins by mass spectrometry or Western blotting to identify novel or context-specific interaction partners.

• Compare interaction profiles under different cellular conditions to elucidate regulatory mechanisms.

RNA immunoprecipitation (RIP) and crosslinking immunoprecipitation (CLIP):

• Apply CCAR2 antibodies in RIP or CLIP protocols to identify RNA molecules directly interacting with CCAR2 or the DBIRD complex.

• Sequence precipitated RNAs to map binding sites and potential regulatory motifs.

• Correlate binding patterns with alternatively spliced exons in (A+T)-rich regions.

Proximity ligation assay (PLA):

• Use CCAR2 antibodies in combination with antibodies against other DBIRD components or RNA processing factors.

• Visualize and quantify protein-protein interactions in situ under different cellular conditions.

• Assess spatial relationships between CCAR2 and the transcriptional/splicing machinery.

These methodologies enable detailed investigation of CCAR2's functional roles in RNA processing and transcriptional regulation, providing insights into how the DBIRD complex modulates gene expression patterns.

What considerations are important when using CCAR2 antibodies in cancer research models?

When employing CCAR2 antibodies in cancer research, several specialized considerations are necessary to generate meaningful and reproducible results:

Tissue-specific expression patterns:

• CCAR2 expression has been documented to increase progressively in gastric carcinoma as the disease advances . When designing experiments, consider tissue-specific baseline expression levels and potential alterations in cancer states.

• Use appropriate control tissues and calibrate antibody dilutions accordingly for comparative analyses between normal and tumor samples.

Subcellular localization changes:

• Monitor potential shifts in CCAR2 localization between nuclear and cytoplasmic compartments in cancer cells compared to normal cells using immunofluorescence or subcellular fractionation followed by Western blotting.

• Changes in localization may indicate altered function or regulation in cancer contexts.

Patient-derived xenograft (PDX) models:

• When using CCAR2 antibodies in PDX models, verify antibody species cross-reactivity to distinguish between human tumor-derived CCAR2 and potential host (typically mouse) homologs.

• Use antibodies with confirmed specificity for human CCAR2 if focusing on the tumor component.

Correlation with clinical parameters:

• When analyzing human cancer samples, correlate CCAR2 expression or localization patterns (detected by IHC) with clinical parameters such as tumor stage, grade, and patient outcomes.

• Consider using tissue microarrays for high-throughput screening across multiple patient samples.

Drug response studies:

• Evaluate changes in CCAR2 expression, localization, or post-translational modifications in response to therapeutic interventions.

• Consider combining CCAR2 antibody-based detection with functional assays to correlate protein changes with biological outcomes.

How do post-translational modifications of CCAR2 affect antibody recognition and experimental design?

Post-translational modifications (PTMs) of CCAR2 can significantly impact antibody recognition and must be considered when designing experiments and interpreting results:

Molecular weight discrepancies:

• The calculated molecular weight of CCAR2 is 103 kDa, but it typically appears at approximately 130 kDa in experimental systems . This discrepancy likely reflects extensive post-translational modifications.

• When analyzing Western blot results, researchers should anticipate this higher apparent molecular weight and not mistake it for non-specific binding.

Phosphorylation-specific detection:

• CCAR2 undergoes phosphorylation under various cellular conditions, which may alter epitope accessibility or antibody binding.

• Consider using phosphorylation-specific antibodies when studying CCAR2 regulation through phosphorylation events.

• Include phosphatase treatments as controls when appropriate to confirm phosphorylation-dependent recognition.

Epitope masking by protein interactions:

• CCAR2's involvement in the DBIRD complex and interactions with other proteins may mask certain epitopes.

• When studying CCAR2 in complex with other proteins, use antibodies targeting different regions of the protein to ensure detection.

• Consider using denaturing conditions in Western blotting to expose epitopes that might be masked in native conformations.

Sample preparation impact:

• Different extraction methods may preserve or disrupt specific PTMs.

• When studying particular modifications, optimize lysis buffers to include appropriate inhibitors (phosphatase inhibitors, deacetylase inhibitors, etc.).

• Consider native versus denaturing conditions based on the specific PTMs being investigated.

Cross-reactivity considerations:

• Some PTMs may create epitopes that cross-react with antibodies targeting other proteins.

• Validate specificity using multiple antibodies targeting different CCAR2 regions or through genetic approaches (knockdown/knockout controls).

What are common challenges when using CCAR2 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with CCAR2 antibodies. Here are methodological solutions to address these issues:

Non-specific bands in Western blotting:

• Challenge: Detection of unexpected bands beyond the anticipated 130 kDa CCAR2 band.

• Solutions: Optimize antibody dilution (start with manufacturer recommendations, typically 1:500-1:2000) ; increase blocking stringency using 5% BSA instead of milk if phospho-epitopes are important; include peptide competition controls to identify specific binding; consider using gradient gels for better resolution of high molecular weight proteins.

Weak or absent signal:

• Challenge: Insufficient detection of CCAR2 despite known expression in the sample.

• Solutions: Verify protein loading with housekeeping controls; reduce sample heating time to prevent potential degradation of large proteins; optimize transfer conditions for high molecular weight proteins; consider longer primary antibody incubation (overnight at 4°C); verify sample preparation methods preserve the epitope of interest.

High background in immunostaining:

• Challenge: Non-specific staining making specific CCAR2 signal difficult to interpret.

• Solutions: Increase blocking time (2+ hours) or concentration; optimize antibody dilution (starting with 1:50-1:200 for IHC-P) ; increase washing duration and number of washes; consider using different blocking agents (normal serum from secondary antibody host species); validate secondary antibody specificity independently.

Inconsistent results between experiments:

• Challenge: Variable detection of CCAR2 across replicate experiments.

• Solutions: Standardize sample collection and processing; aliquot antibodies to avoid freeze-thaw cycles; maintain consistent incubation times and temperatures; document lot numbers of antibodies used and note any performance variations.

How should researchers validate CCAR2 antibody specificity in their experimental systems?

Comprehensive validation of CCAR2 antibody specificity is crucial for generating reliable research data. The following methodological approaches are recommended:

Genetic validation approaches:

• Implement CCAR2 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9) controls to confirm signal specificity.

• Compare signal intensity between wild-type and CCAR2-depleted samples using Western blotting, immunofluorescence, or immunohistochemistry.

• Expect proportional reduction in signal corresponding to knockdown efficiency or complete absence in knockout systems.

Peptide competition assays:

• Pre-incubate the CCAR2 antibody with excess immunizing peptide before application to samples.

• A specific antibody will show significantly reduced or eliminated signal when pre-absorbed with its target peptide.

• This method is particularly valuable for polyclonal antibodies that might recognize multiple epitopes.

Multiple antibody validation:

• Use different antibodies targeting distinct CCAR2 epitopes and compare detection patterns.

• Consistent results across antibodies raised against different regions of CCAR2 provide strong evidence for specificity.

• Consider using both monoclonal and polyclonal antibodies as complementary approaches.

Recombinant expression systems:

• Overexpress tagged CCAR2 (full-length or fragments) and confirm detection by both the CCAR2 antibody and tag-specific antibodies.

• This approach is particularly useful for mapping the specific epitope recognized by monoclonal antibodies.

• Can also help distinguish between isoforms if expressed individually.

Mass spectrometry verification:

• Immunoprecipitate CCAR2 using the antibody in question and analyze by mass spectrometry.

• Identification of CCAR2 peptides as predominant components in the immunoprecipitated material confirms specificity.

• This approach can also identify co-precipitating proteins and potential complex components.

How can CCAR2 monoclonal antibodies be adapted for use in high-throughput screening approaches?

CCAR2 monoclonal antibodies can be effectively integrated into high-throughput screening platforms through several methodological adaptations:

Automated immunofluorescence platforms:

• Adapt CCAR2 immunostaining protocols for 96- or 384-well plate formats compatible with automated microscopy systems.

• Optimize fixation, permeabilization, and antibody incubation times for shorter processing while maintaining signal quality.

• Implement dual staining with markers of cell cycle phases to correlate CCAR2 expression or localization with cell cycle progression.

• Develop algorithms for automated image analysis to quantify CCAR2 signal intensity, subcellular localization, and co-localization with other proteins of interest.

Protein microarray applications:

• Utilize CPTC-KIAA1967-1 or other validated monoclonal antibodies suited for microarray applications .

• Develop reverse-phase protein arrays (RPPAs) to rapidly assess CCAR2 expression across multiple samples simultaneously.

• Implement antibody arrays to evaluate CCAR2 interaction partners under various treatment conditions.

• Consider bead-based multiplexed assays to simultaneously detect CCAR2 alongside other proteins of interest in signaling pathways.

Flow cytometry and cell sorting:

• Adapt CCAR2 antibodies for intracellular flow cytometry by optimizing fixation and permeabilization protocols.

• Combine with fluorescent cell cycle indicators to correlate CCAR2 expression with cell cycle stages across large cell populations.

• Utilize fluorescence-activated cell sorting (FACS) with CCAR2 antibodies to isolate cell populations with different CCAR2 expression levels for downstream analysis.

High-content screening in drug discovery:

• Develop CCAR2 antibody-based assays to screen compound libraries for modulators of CCAR2 expression, localization, or post-translational modifications.

• Implement multiplexed detection systems to simultaneously assess CCAR2 and downstream cellular responses.

• Establish dose-response protocols with automated liquid handling systems for efficient screening of candidate compounds.

What is the potential for using CCAR2 antibodies in combination with other therapeutic monoclonal antibodies in cancer research?

The integration of CCAR2 antibodies with therapeutic monoclonal antibodies presents promising opportunities for advancing cancer research:

Mechanism of action studies:

• Use CCAR2 antibodies as research tools to investigate how therapeutic monoclonal antibodies affect CCAR2-mediated pathways in cancer cells .

• Employ immunofluorescence or immunohistochemistry to assess changes in CCAR2 expression or localization following treatment with therapeutic antibodies.

• Implement time-course studies to map the temporal relationship between therapeutic antibody administration and CCAR2-associated cellular responses.

Resistance mechanism investigation:

• Apply CCAR2 antibodies to compare expression and localization patterns between treatment-responsive and treatment-resistant cancer models.

• Investigate whether CCAR2-related pathways contribute to acquired resistance to therapeutic monoclonal antibodies.

• Use co-immunoprecipitation with CCAR2 antibodies to identify potential interaction partners that might influence therapeutic efficacy.

Combination therapy development:

• Evaluate whether CCAR2 pathway modulation enhances the efficacy of existing therapeutic antibodies.

• Investigate potential synergistic or antagonistic effects between CCAR2-targeting approaches and therapeutic monoclonal antibodies.

• Utilize CCAR2 antibodies in preclinical models to identify optimal timing and sequencing of combination treatments.

Biomarker development:

• Assess CCAR2 expression, localization, or post-translational modifications as potential predictive biomarkers for therapeutic antibody response.

• Develop standardized protocols for CCAR2 immunostaining in patient samples that could be integrated into clinical trial companion diagnostics.

• Correlate CCAR2 status with clinical outcomes in patients receiving therapeutic monoclonal antibodies.

It's important to note that this research application remains experimental, and researchers should design rigorous validation studies when exploring the relationship between CCAR2 biology and therapeutic antibody efficacy .

How might emerging antibody technologies advance CCAR2 research beyond current capabilities?

Several cutting-edge antibody technologies have the potential to significantly enhance CCAR2 research:

Single-domain antibodies and nanobodies:

• Developing CCAR2-specific nanobodies could enable access to epitopes inaccessible to conventional antibodies due to their smaller size (~15 kDa vs. ~150 kDa).

• Their enhanced tissue penetration makes them valuable for in vivo imaging of CCAR2 in tumor models.

• Their stability allows for more robust intracellular expression as "intrabodies" to monitor or modulate CCAR2 function in living cells.

Bispecific antibodies for co-localization studies:

• Engineering bispecific antibodies targeting CCAR2 and other DBIRD complex components would enable direct visualization of protein interactions.

• These tools could help map the spatial and temporal dynamics of CCAR2-containing complexes during transcription and RNA processing.

• They could also be adapted for selective immunoprecipitation of specific CCAR2-containing subcomplexes.

Antibody-DNA conjugates for spatial genomics:

• Conjugating CCAR2 antibodies with DNA barcodes would enable spatial mapping of CCAR2 distribution in relation to specific genomic loci.

• This approach could advance understanding of how CCAR2 and the DBIRD complex regulate gene expression in different chromatin contexts.

• Combined with single-cell technologies, this could reveal cell-to-cell variation in CCAR2 function within heterogeneous tumor samples.

Recombinant antibody engineering:

• Developing recombinant CCAR2 antibody fragments with enhanced specificity for different protein domains would enable more precise functional studies.

• Site-specific conjugation techniques could produce homogeneous antibody-fluorophore conjugates with improved signal-to-noise ratios for imaging applications.

• Humanized CCAR2 antibodies could potentially bridge the gap between research tools and therapeutic development.

What are the key unresolved questions about CCAR2 that could be addressed with improved antibody tools?

Several critical questions in CCAR2 biology remain unanswered and could be addressed with advanced antibody-based approaches:

Isoform-specific functions:

• Development of isoform-specific CCAR2 antibodies would enable differential detection of the reported CCAR2 isoforms .

• These tools could help determine whether different isoforms have distinct subcellular localizations, interaction partners, or functions.

• Research question: Do CCAR2 isoforms play different roles in normal versus malignant cells?

Post-translational modification mapping:

• Generation of modification-specific antibodies (phospho, acetyl, etc.) would enable detailed mapping of CCAR2 regulation.

• These tools could help identify which modifications correlate with various cellular states or disease progression.

• Research question: How do specific post-translational modifications alter CCAR2's role in the DBIRD complex and affect RNA processing?

Dynamic complex assembly:

• Using proximity labeling approaches with CCAR2 antibodies could illuminate the temporal sequence of DBIRD complex assembly.

• Super-resolution microscopy with specialized antibody probes could visualize CCAR2-containing complexes at near-molecular resolution.

• Research question: What is the stepwise process of DBIRD complex assembly and how does CCAR2 contribute to its structural integrity?

Cancer-specific alterations:

• Developing antibodies capable of distinguishing between normal and cancer-associated forms of CCAR2 could enhance diagnostic applications.

• Tools to detect cancer-specific interactions or modifications might reveal new therapeutic vulnerabilities.

• Research question: Does CCAR2 undergo cancer-specific alterations that contribute to disease progression or treatment resistance?

These unresolved questions represent significant opportunities for researchers to advance understanding of CCAR2 biology through innovative antibody-based approaches, potentially leading to new diagnostic or therapeutic strategies targeting CCAR2-mediated pathways.