BTRC Antibody

What is BTRC and why is it significant in research?

BTRC (beta-Transducin Repeat Containing) is a member of the F-box protein family characterized by an approximately 40 amino acid motif called the F-box. This protein functions as a critical component of SCF (SKP1-cullin-F-box) ubiquitin protein ligase complexes, which are responsible for phosphorylation-dependent ubiquitination of target proteins . BTRC plays a particularly important role in the Wnt signaling pathway, making it a valuable target for research into developmental processes and disease mechanisms . The protein's involvement in protein ubiquitination and degradation pathways positions it as a key regulatory element in multiple cellular processes including cell cycle regulation, signal transduction, and protein quality control. Researchers typically study BTRC to understand its role in normal cellular physiology and its dysregulation in various pathological conditions.

How do I select the appropriate BTRC antibody for my research application?

Selecting the optimal BTRC antibody requires careful consideration of multiple experimental parameters. First, determine your specific application needs (Western blotting, immunohistochemistry, immunofluorescence, flow cytometry, or ELISA) as different antibodies exhibit varying performance across applications . Next, consider the species reactivity requirements - available antibodies demonstrate reactivity to human, mouse, and/or rat BTRC . The choice between monoclonal and polyclonal antibodies should be based on your specific experimental needs: monoclonals (e.g., clone 4C5D8, 3A2F11A12, or AB02/4E2) offer high specificity and reproducibility, while polyclonals provide broader epitope recognition .

Pay particular attention to the binding specificity region, as different antibodies target distinct amino acid sequences within BTRC (e.g., AA 24-151, AA 50-340, AA 17-52) . This is especially important when investigating specific domains or when potential cross-reactivity with related proteins is a concern. Finally, examine validation data provided by manufacturers, looking for evidence of specificity such as the ~55 kDa band detected in HepG2 cell lysates by the AB02/4E2 clone .

What are the optimal protocols for using BTRC antibodies in Western Blotting?

For optimal Western blotting results with BTRC antibodies, follow this methodological approach based on validated protocols. Begin sample preparation by extracting proteins under conditions that preserve BTRC integrity - typically RIPA buffer supplemented with protease and phosphatase inhibitors. Separate 20-40 μg of protein per lane using 8-10% SDS-PAGE gels to achieve optimal resolution of BTRC, which appears at approximately 55 kDa in HepG2 cell lysates .

Transfer proteins to PVDF membranes (preferred over nitrocellulose for BTRC) using semi-dry transfer systems at 15V for 30 minutes or wet transfer at 100V for 60 minutes. Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature. For primary antibody incubation, dilute BTRC antibodies according to manufacturer recommendations - typically 1:1000 for Western blotting with the AB02/4E2 clone . Incubate overnight at 4°C with gentle agitation.

Wash membranes thoroughly (4 × 5 minutes with TBST) before applying appropriate species-specific secondary antibodies conjugated to HRP. Include positive controls (HepG2 lysates) and negative controls (lysates from cells with BTRC knockdown) to validate specificity. For signal development, enhanced chemiluminescence systems provide excellent sensitivity for BTRC detection.

How should I optimize immunohistochemistry procedures when using BTRC antibodies?

Optimizing immunohistochemistry (IHC) procedures for BTRC antibodies requires careful attention to tissue processing, antigen retrieval, and detection methods. Begin with proper fixation - 10% neutral buffered formalin for 24-48 hours is recommended, followed by paraffin embedding. Cut sections at 4-5 μm thickness for optimal antibody penetration and signal-to-noise ratio.

Antigen retrieval is critical for BTRC detection in fixed tissues. Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-100°C for 20 minutes typically yields superior results. For BTRC antibodies, particularly those targeting amino acids 24-151 or 50-340, a cooling period of 20 minutes following heat treatment enhances epitope accessibility .

For the immunostaining procedure, block endogenous peroxidase activity with 3% hydrogen peroxide, followed by protein blocking with 5% normal serum. Apply primary BTRC antibodies at optimized dilutions (typically 1:100 to 1:500) and incubate in a humidified chamber at 4°C overnight. Multiple BTRC antibodies have been validated for IHC applications, including both polyclonal and monoclonal variants targeting different epitopes . After thorough washing, apply appropriate detection systems - polymer-based detection methods generally provide superior signal-to-noise ratios compared to traditional avidin-biotin complexes for BTRC visualization.

What approaches are recommended for validating BTRC antibody specificity?

Validating BTRC antibody specificity requires a multi-faceted approach combining molecular, cellular, and analytical techniques. First, perform peptide competition assays by pre-incubating the antibody with excess purified BTRC peptide (matching the immunogen sequence) before application to samples. A significant reduction in signal indicates specificity for the target epitope. For BTRC antibodies generated against specific regions (e.g., AA 50-340 or AA 24-151), this validation step is particularly informative .

Employ genetic approaches using BTRC-knockout or knockdown models. Compare antibody reactivity between wild-type samples and those with reduced/absent BTRC expression. The absence of signal in knockout samples provides compelling evidence of specificity. Western blot analysis should demonstrate bands at the expected molecular weight (~55 kDa for BTRC) , while showing appropriate signal reduction in knockdown samples.

Cross-validation using multiple antibodies targeting different BTRC epitopes provides additional confidence. For instance, compare results from antibodies targeting the N-terminal region (AA 17-52) versus the central domain (AA 50-340) . Concordant results across different antibodies suggest true BTRC detection rather than cross-reactivity. When possible, mass spectrometry analysis of immunoprecipitated proteins can provide definitive confirmation of antibody specificity, identifying BTRC-specific peptides from the immunoprecipitated material.

How can BTRC antibodies be used to study the Wnt signaling pathway?

BTRC antibodies serve as powerful tools for investigating the Wnt signaling pathway due to BTRC's involvement as an E3 ubiquitin ligase component that targets key Wnt pathway elements. For studying BTRC's role in β-catenin regulation, implement co-immunoprecipitation (Co-IP) experiments using BTRC antibodies to capture protein complexes. This approach can reveal dynamic interactions between BTRC and phosphorylated β-catenin under different Wnt signaling states .

For visualizing the spatial distribution of BTRC in relation to other Wnt pathway components, employ immunofluorescence (IF) using antibodies validated for this application, such as the monoclonal 3A2F11A12 antibody . Combine BTRC antibody labeling with antibodies against other Wnt pathway proteins (e.g., β-catenin, GSK3β) to assess colocalization under different signaling conditions. Confocal microscopy analysis can reveal subcellular compartment-specific interactions relevant to Wnt signaling regulation.

To quantitatively assess how Wnt pathway activation affects BTRC-mediated protein degradation, implement pulse-chase experiments combined with immunoprecipitation using BTRC antibodies. This methodology allows for tracking the kinetics of BTRC-dependent substrate degradation. Additionally, chromatin immunoprecipitation (ChIP) assays using antibodies against BTRC and TCF/LEF transcription factors can reveal how BTRC-mediated protein turnover influences transcriptional regulation of Wnt target genes.

What techniques can be used to investigate BTRC-mediated protein ubiquitination?

Investigating BTRC-mediated protein ubiquitination requires specialized techniques that capture this transient post-translational modification. In vitro ubiquitination assays represent a powerful approach, where purified components (E1, E2, BTRC-containing E3 complex, ubiquitin, ATP, and substrate) are combined, followed by detection of ubiquitinated products via Western blotting using both BTRC antibodies and ubiquitin-specific antibodies.

For cellular systems, implement tandem ubiquitin binding entity (TUBE) pulldowns coupled with BTRC immunoblotting. This technique selectively enriches for ubiquitinated proteins from cell lysates, after which BTRC antibodies can detect BTRC-associated ubiquitinated substrates. Cell-based ubiquitination assays using HA-tagged ubiquitin expression combined with immunoprecipitation using BTRC antibodies (such as the polyclonal antibody targeting AA 50-340) provide another effective approach. This methodology allows for the isolation of BTRC-substrate complexes and subsequent detection of ubiquitination status.

Mass spectrometry analysis following BTRC immunoprecipitation can provide comprehensive identification of BTRC-associated ubiquitinated proteins and mapping of specific ubiquitination sites. For temporal resolution, implement cycloheximide chase assays combined with BTRC immunoblotting to track the degradation kinetics of BTRC substrates, revealing the functional consequences of BTRC-mediated ubiquitination on protein stability.

What methods can researchers use to study BTRC in different cellular compartments?

Studying BTRC localization across cellular compartments requires complementary approaches that provide both spatial and biochemical information. Subcellular fractionation combined with Western blotting using specific BTRC antibodies (such as the monoclonal AB02/4E2 clone) allows for biochemical quantification of BTRC distribution across nuclear, cytoplasmic, membrane, and organelle fractions. This approach provides quantitative assessment of compartment-specific BTRC levels.

For high-resolution spatial analysis, implement immunofluorescence microscopy using BTRC antibodies validated for IF applications, such as the monoclonal 3A2F11A12 or polyclonal antibodies targeting amino acids 50-340 . Co-staining with organelle-specific markers (e.g., lamin B for nuclear envelope, calnexin for ER, GM130 for Golgi) enables precise localization of BTRC relative to cellular structures. Super-resolution microscopy techniques like STORM or STED can provide nanoscale resolution of BTRC distribution within compartments.

Proximity ligation assay (PLA) using BTRC antibodies paired with antibodies against compartment-specific interacting partners offers a powerful approach to visualizing and quantifying compartment-specific BTRC interactions. For dynamic tracking of BTRC translocation between compartments, combine live-cell imaging of fluorescently-tagged BTRC with fixed-cell immunostaining using BTRC antibodies to validate the physiological relevance of observed trafficking patterns.

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

The choice between monoclonal and polyclonal BTRC antibodies significantly impacts experimental outcomes, with each offering distinct advantages. Monoclonal BTRC antibodies, such as clones 4C5D8 (targeting AA 24-151), 3A2F11A12, and AB02/4E2 (targeting AA 52-354), provide high specificity for discrete epitopes, resulting in exceptional batch-to-batch consistency and reduced background signal . These properties make monoclonals particularly valuable for quantitative applications requiring reproducible results across experiments.

Polyclonal BTRC antibodies, such as those targeting amino acids 50-340 or the N-terminal region, recognize multiple epitopes within the target region, providing enhanced sensitivity through signal amplification . This characteristic makes polyclonals advantageous for detecting low-abundance BTRC expression or post-translationally modified forms. The multi-epitope recognition also increases robustness against epitope masking that might occur due to protein-protein interactions or conformational changes.

For applications requiring high specificity such as distinguishing between BTRC paralogs or isoforms, monoclonal antibodies typically provide superior discrimination. Conversely, for applications focused on capturing the total BTRC population or when target conformations may vary, polyclonal antibodies often demonstrate superior performance.

What factors might affect BTRC antibody cross-reactivity across species?

BTRC antibody cross-reactivity across species is influenced by multiple factors that researchers must consider when designing cross-species experiments. Sequence homology between species represents the primary determinant of cross-reactivity. Carefully examine the amino acid sequence conservation in the epitope region targeted by your antibody. For instance, antibodies targeting the highly conserved AA 50-340 region of BTRC often demonstrate cross-reactivity among human, mouse, and rat samples .

Epitope accessibility varies between species due to differences in post-translational modifications, protein-protein interactions, or tertiary structure. These variations can mask epitopes despite sequence conservation. When validating cross-species reactivity, implement appropriate positive controls from each target species alongside human samples as reference standards.

Processing methods significantly impact cross-reactivity outcomes. Fixation protocols, particularly for IHC applications, may differentially affect epitope preservation across species. Modified antigen retrieval methods may be necessary when applying human-validated BTRC antibodies to rodent tissues. For optimal cross-species applications, consider antibodies specifically validated across multiple species, such as those documented to react with human, mouse, and rat BTRC .

How should contradictory results from different BTRC antibodies be addressed?

Contradictory results from different BTRC antibodies require systematic investigation through multiple analytical approaches. Begin by examining the epitope specificity of each antibody. Antibodies targeting different regions of BTRC (e.g., N-terminal AA 17-52 versus central domain AA 50-340) may yield different results if post-translational modifications, protein interactions, or conformational changes affect epitope accessibility in your experimental system .

Implement validation experiments using orthogonal techniques. If Western blotting results conflict between antibodies, verify BTRC expression using RT-qPCR to quantify mRNA levels. For contradictory subcellular localization patterns, compare immunofluorescence results with biochemical fractionation experiments. Supporting evidence from non-antibody-based methods can help determine which antibody provides more accurate results.

Consider isoform specificity as a source of discrepancy. BTRC exists in multiple isoforms, and antibodies may preferentially detect specific variants. Review the literature and sequence databases to identify known BTRC isoforms and their expression patterns in your experimental system. Antibodies targeting common regions will detect multiple isoforms, while those against isoform-specific sequences will yield more selective patterns.

When reporting conflicting results, transparently document all antibodies used (including catalog numbers and clones such as 4C5D8, 3A2F11A12, or AB02/4E2), experimental conditions, and validation steps undertaken . This approach ensures reproducibility and facilitates the collective advancement of BTRC research.

How can BTRC antibodies be used to investigate its role in cancer pathways?

BTRC antibodies provide valuable tools for investigating the protein's complex roles in cancer pathways through multiple experimental approaches. For studying BTRC expression patterns across cancer types, implement tissue microarray (TMA) analysis using immunohistochemistry with validated BTRC antibodies such as those targeting amino acids 50-340 or 24-151 . This approach allows for high-throughput screening of BTRC expression across multiple tumor samples and correlation with clinical parameters.

To investigate the mechanistic role of BTRC in regulating cancer-related proteins, employ co-immunoprecipitation using BTRC antibodies followed by mass spectrometry to identify novel cancer-relevant interaction partners. This approach can reveal previously unknown substrates of BTRC-mediated ubiquitination that may contribute to oncogenesis or tumor suppression. For confirming specific interactions, reverse co-IP experiments using antibodies against suspected binding partners can validate BTRC associations.

Chromatin immunoprecipitation sequencing (ChIP-seq) combined with BTRC antibodies can reveal genome-wide binding patterns of BTRC at promoter regions of cancer-related genes. This approach is particularly relevant given BTRC's role in regulating transcription factors through the Wnt signaling pathway . For functional studies, combine BTRC knockdown/overexpression with immunoblotting using BTRC antibodies to track changes in the stability of cancer-relevant proteins like β-catenin, providing insights into how BTRC dysregulation contributes to oncogenic signaling.

What are the best practices for using BTRC antibodies in high-throughput screens?

Implementing BTRC antibodies in high-throughput screening applications requires careful optimization to ensure reproducibility, sensitivity, and specificity across large sample sets. For plate-based immunoassays, start by validating antibody performance using a dose-response curve of recombinant BTRC protein to establish detection limits and linear range. Select antibodies with demonstrated specificity, such as well-characterized monoclonal antibodies (clones 3A2F11A12 or 4C5D8) that minimize cross-reactivity in complex samples .

Develop robust automated protocols for high-throughput screening platforms. For cell-based screens, optimize fixation methods, antibody concentrations, incubation times, and washing procedures to maximize signal-to-noise ratios while maintaining throughput. Implement automated image acquisition and analysis algorithms that can reliably quantify BTRC levels, localization patterns, or interaction dynamics across thousands of samples.

Quality control is critical for high-throughput applications. Implement Z-factor calculations to assess assay quality, with values above 0.5 indicating suitable assay performance. Include internal controls on each plate to monitor plate-to-plate and day-to-day variability. For multiplexed screens, carefully validate that BTRC antibodies perform consistently when combined with antibodies against other targets, checking for potential interference effects.

How can BTRC antibodies be integrated with advanced imaging techniques?

Integration of BTRC antibodies with advanced imaging techniques enables unprecedented insights into protein dynamics, interactions, and functions in cellular contexts. For super-resolution microscopy applications (STORM, PALM, STED), select BTRC antibodies with high specificity and appropriate fluorophore conjugation. Monoclonal antibodies like 3A2F11A12, validated for immunofluorescence applications, provide the precision required for nanoscale resolution imaging . When implementing direct immunofluorescence approaches, verify that fluorophore conjugation does not compromise antibody binding characteristics or specificity.

For live-cell imaging applications, combine traditional fixed-cell immunostaining using BTRC antibodies with genetically encoded fluorescent protein fusions to validate physiological relevance of observed dynamics. This dual approach provides complementary insights, with antibody staining offering endogenous protein visualization and genetic fusions enabling real-time tracking.

Expand spatial context through multiplexed imaging approaches. Implement sequential immunofluorescence methods using BTRC antibodies in combination with antibodies against interaction partners, signaling pathway components, or cellular structure markers. Cyclic immunofluorescence or mass cytometry (CyTOF) techniques allow simultaneous visualization of dozens of proteins, positioning BTRC within its complex signaling network.

For correlative light and electron microscopy (CLEM), use BTRC antibodies conjugated to both fluorescent tags and electron-dense particles. This approach bridges the resolution gap between fluorescence microscopy and electron microscopy, providing both spatial context and ultrastructural details of BTRC localization. When implementing these advanced techniques, include appropriate controls to distinguish specific from non-specific signals, particularly as signal amplification methods can exacerbate background issues.