RCBTB2 Antibody
Description
Definition and Biological Context
The RCBTB2 Antibody is a polyclonal rabbit IgG antibody designed to target the regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 2 (RCBTB2), a member of the RCC1-related guanine nucleotide exchange factor (GEF) family. This protein facilitates nucleotide exchange for the Ras-related GTPase Ran, playing a critical role in mitotic regulation and chromatin dynamics . The antibody is widely utilized in molecular biology research for detecting RCBTB2 in human, mouse, and rat samples via techniques such as Western blotting (WB), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) .
Detection Methods
The antibody is validated for:
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Western Blotting: Detects RCBTB2 in thymus tissues of mice and rats at dilutions of 1:1000–1:4000 .
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Immunofluorescence: Localizes RCBTB2 in cellular compartments, aiding studies of mitotic spindle assembly .
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ELISA: Quantifies RCBTB2 in biofluids (e.g., tissue homogenates) with a sensitivity of 0.124 ng/mL .
Research Findings
Published Studies highlight RCBTB2’s role in:
Product Specs
Target Background
- Reduced expression of RCBTB2 in prostate cancer, coupled with differences in variant isoforms between normal and cancerous prostate tissues, suggests its crucial role or proximity to a gene involved in prostate cancer development. PMID: 12115502
Q&A
What is RCBTB2 and why is it studied in molecular research?
RCBTB2 (Regulator of Chromosome Condensation and BTB Domain-Containing Protein 2) is a member of the RCC1-related GEF family. The N-terminal half of its amino acid sequence shows similarity to the regulator of chromosome condensation RCC1, which acts as a guanine nucleotide exchange factor (GEF) protein for the Ras-related GTPase Ran . This protein is also known by several synonyms including CHC1-L, RLG, and RCC1-like G exchanging factor . The canonical human protein consists of 551 amino acid residues with a molecular mass of approximately 60.3 kDa, though the observed molecular weight in laboratory conditions is typically 58-60 kDa .
Research interest in RCBTB2 stems from its widespread expression pattern across multiple tissue types and its potential roles in cellular regulation processes . Studies have implicated RCBTB2 in various biological processes, including roles in spermatogenesis in humans and mice, as indicated by published research using RCBTB2 antibodies .
What applications are commonly supported by commercial RCBTB2 antibodies?
RCBTB2 antibodies support multiple research applications, with varying degrees of validation across different experimental methodologies:
When selecting an RCBTB2 antibody, researchers should prioritize reagents that have been validated specifically for their intended application, as performance can vary significantly between experimental contexts .
What species reactivity should be considered when selecting RCBTB2 antibodies?
Most commercial RCBTB2 antibodies demonstrate reactivity with human samples, while reactivity with other species varies by product:
When working with non-human models, verify the specific sequence homology and empirical validation data. For example, some antibodies demonstrate documented cross-reactivity with samples from mouse (96%), rat (96%), xenopus laevis (83%), and bovine (96%) , reflecting the evolutionary conservation of RCBTB2 protein sequences across these species.
How should I select between polyclonal and monoclonal RCBTB2 antibodies?
The choice between polyclonal and monoclonal RCBTB2 antibodies depends on your experimental requirements:
For initial characterization studies, a polyclonal antibody may provide better detection sensitivity, while monoclonal antibodies offer advantages for standardized assays requiring high reproducibility and specificity . Polyclonal RCBTB2 antibodies targeting different regions (such as N-terminal regions, internal regions, or specific amino acid ranges) are more widely available commercially than monoclonal variants .
What are the optimal dilution protocols for different RCBTB2 antibody applications?
Optimization of antibody dilution is critical for balancing signal strength with background reduction:
While these ranges provide starting points, antibody titration experiments are essential for determining optimal concentration for specific sample types and experimental conditions. As noted in technical documentation: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and "Sample-dependent, check data in validation data gallery" .
What is the recommended storage and handling protocol to maintain RCBTB2 antibody performance?
Proper storage and handling significantly impact antibody shelf-life and performance:
Most manufacturers recommend avoiding repeated freeze-thaw cycles to preserve antibody integrity. For example, one product specifies: "Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20°C storage" , while another notes: "Aliquot and store at -20°C. Avoid repeated freeze/thaw cycles" .
How can I verify the specificity of RCBTB2 antibodies in my experimental system?
Verifying antibody specificity requires multi-parameter validation:
Published studies using RCBTB2 antibodies provide valuable references for expected results. For instance, RCBTB2 antibodies have been used successfully in research examining spermatogenesis, ovarian cancer, and HepG2 hepatoma cells , providing benchmarks for comparison.
What are common causes of inconsistent results when working with RCBTB2 antibodies?
When troubleshooting variable or unexpected results with RCBTB2 antibodies, consider these common sources of error:
For immunohistochemistry applications, pay particular attention to the fixation method, as this can significantly impact epitope accessibility and RCBTB2 antibody performance .
How does subcellular localization affect RCBTB2 detection methodologies?
RCBTB2 has been reported to localize to cytoplasmic vesicles, secretory vesicles, and the acrosome (mainly in the acrosomal cap region) . This localization pattern necessitates specific methodological considerations:
| Technique | Methodological Considerations | Optimization Strategies |
|---|---|---|
| Immunofluorescence | - Cell permeabilization crucial - Co-localization with vesicular markers | - Try different permeabilization agents (Triton X-100, saponin) - Combine with markers for specific vesicle types |
| Subcellular Fractionation | - Enrichment of vesicular fractions - Potential loss during purification | - Use differential centrifugation protocols optimized for vesicles - Include markers for different cellular compartments |
| Electron Microscopy | - Precise localization within vesicular structures | - Immunogold labeling for high-resolution localization |
| Biochemical Extractions | - May require specialized buffers for vesicle disruption | - Test detergent-based and mechanical disruption methods |
Understanding the vesicular nature of RCBTB2 localization can help explain patterns observed in immunostaining and improve detection strategies for this protein in different cellular contexts .
How do different epitope targets affect RCBTB2 antibody performance?
Commercial RCBTB2 antibodies target various regions of the protein, which can significantly affect detection characteristics:
The choice of epitope region can affect antibody performance under different experimental conditions. For example, antibodies targeting the N-terminal region may be more sensitive to proteolytic processing events, while those targeting internal regions might better withstand denaturation in Western blotting .
What methodological approaches can optimize RCBTB2 detection in various experimental systems?
Optimizing RCBTB2 detection requires considering both the protein's characteristics and experimental context:
For challenging applications, consider using multiple antibodies targeting different epitopes to validate findings, particularly when studying potential protein-protein interactions or post-translational modifications .
How can RCBTB2 antibodies be employed in investigating protein-protein interactions?
Investigating RCBTB2's interaction network requires specialized methodological approaches:
The BTB (POZ) domain in RCBTB2 suggests potential protein-protein interaction capabilities, as this domain is known to mediate such interactions in other proteins. Antibodies targeting this domain may be particularly useful for investigating RCBTB2's protein interaction network .
What are the methodological considerations for studying RCBTB2 in different model organisms?
Research across different model organisms requires careful consideration of sequence conservation and antibody cross-reactivity:
When working with non-human models, preliminary validation experiments are essential. Western blotting with tissue lysates from the target organism can confirm antibody cross-reactivity before proceeding to more complex applications .
How can RCBTB2 antibodies be used to investigate the protein's role in disease mechanisms?
RCBTB2 antibodies have been employed in multiple disease-related research contexts:
When designing disease-related studies, consider tissue microarrays for screening RCBTB2 expression across multiple patient samples, and multiplex staining approaches to correlate RCBTB2 levels with other disease markers . The subcellular localization in vesicular structures may provide clues to RCBTB2's potential roles in secretory pathways relevant to disease progression .
