BRG3 Antibody

What is BTG3 protein and what are its main functions?

BTG3 (B-cell translocation gene 3) is a member of the BTG/TOB family of antiproliferative proteins. Its primary function involves regulating cell cycle progression, specifically by impeding the transition from G0/G1 to S phase when overexpressed . The protein is ubiquitously expressed in human tissues, with notably higher expression observed in the ventricular zone of the developing central nervous system, as well as in reproductive and lymphoid tissues including ovary, testis, prostate, thymus, and lung . BTG3 functions as a negative regulator of cell proliferation, potentially playing important roles in development, differentiation, and tumor suppression. The protein is known by several alternative names including "abundant in neuroepithelium area protein" (ANA) and protein Tob5, reflecting its varied tissue distribution and biological roles .

What is the difference between BTG3 and BRG3?

Despite similar abbreviations that sometimes cause confusion, BTG3 and BRG3 are distinct proteins with different functions:

BRG3 has been studied in plant biology contexts, particularly regarding its role in tomato ripening processes. Research has shown that BRG3 can undergo persulfidation (addition of sulfhydryl groups) at specific cysteine residues (Cys 206 and Cys 212), which reduces its ubiquitination activity and affects its interactions with transcription factors like WRKY71 . This mechanism plays a role in delaying tomato fruit ripening through complex regulatory pathways involving protein-protein interactions and transcriptional regulation.

What are the common applications of BTG3 antibodies in research?

BTG3 antibodies serve various research applications, with the most common being:

• Western Blotting (WB): BTG3 antibodies are frequently used at dilutions of approximately 1:1000 for detecting endogenous levels of BTG3 protein in cell and tissue lysates . This application allows researchers to determine protein expression levels and validate knockout or overexpression models.

• Immunohistochemistry-Paraffin (IHC-P): At dilutions ranging from 1:10 to 1:50, BTG3 antibodies can visualize the spatial distribution of BTG3 protein in fixed, paraffin-embedded tissue sections . This application is particularly valuable for studying expression patterns in developmental contexts or disease states.

• Flow Cytometry: Though less commonly referenced in the provided materials, properly validated BTG3 antibodies can be used to measure BTG3 expression in individual cells within heterogeneous populations .

• Mechanistic Studies: BTG3 antibodies are essential tools for investigating the protein's role in cell cycle regulation, particularly regarding the G0/G1 to S phase transition .

• Protein Interaction Studies: Through techniques like co-immunoprecipitation, BTG3 antibodies help identify binding partners and protein complexes that regulate or are regulated by BTG3.

The choice of application should determine which specific BTG3 antibody format is selected, as not all antibodies perform equally across different experimental platforms.

How are BTG3 antibodies validated for research applications?

Validation of BTG3 antibodies follows comprehensive protocols aligned with the "five pillars" recommendations from the International Working Group for Antibody Validation (IWGAV), addressing the broader scientific reproducibility challenges . Proper validation typically includes:

• Genetic Strategies:

  • CRISPR-Cas9 gene editing: Creating BTG3 knockouts to confirm antibody specificity

  • siRNA knockdown: Reducing BTG3 expression to verify corresponding signal reduction

• Independent Antibody Validation: Using different antibodies targeting distinct epitopes of BTG3 to confirm consistent results

• Orthogonal Validation: Correlating protein detection with mRNA expression data

• Expression Validation: Testing in cell lines with known BTG3 expression profiles (e.g., HeLa cells, which are documented to express BTG3)

• Biochemical Validation:

  • Immunoprecipitation followed by mass spectrometry

  • Western blotting to confirm expected molecular weight

• Application-Specific Validation: For flow cytometry applications, testing on relevant cell lines or primary cells known to express BTG3 under appropriate conditions

This multi-faceted approach ensures that antibodies specifically recognize BTG3 protein without cross-reactivity to other proteins, thereby generating reliable and reproducible research results.

What are the optimal storage conditions for BTG3 antibodies?

Proper storage of BTG3 antibodies is crucial for maintaining their specificity and activity over time. Based on manufacturer recommendations, researchers should follow these guidelines:

• Short-term Storage: Maintain refrigerated at 2-8°C for up to 2 weeks . This temperature range prevents protein denaturation while allowing convenient access for ongoing experiments.

• Long-term Storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles . Repeated freezing and thawing significantly diminishes antibody activity and specificity over time.

• Aliquoting Strategy: Upon receipt, divide the antibody into small single-use aliquots before freezing to minimize freeze-thaw cycles. This practice is particularly important for polyclonal antibodies, such as the rabbit polyclonal BTG3 antibodies described in the search results.

• Buffer Considerations: Most commercial BTG3 antibodies are supplied in phosphate-buffered saline (PBS) with 0.09% (W/V) sodium azide as a preservative . This formulation helps maintain antibody stability during storage.

• Reconstitution Practices: For lyophilized antibodies, reconstitute according to manufacturer instructions immediately prior to use, and store any unused reconstituted antibody following the guidelines above.

Adhering to these storage practices ensures optimal antibody performance and contributes significantly to experimental reproducibility across different research projects and timeframes.

How can researchers ensure specificity when using BTG3 antibodies in complex tissue samples?

Ensuring antibody specificity in complex tissue samples requires a multi-faceted approach that combines careful experimental design with appropriate controls:

• Epitope Mapping Validation: Select antibodies targeting well-characterized epitopes in the central region of human BTG3 (amino acids 100-129) . This region tends to have lower homology with related proteins, reducing cross-reactivity concerns.

• Cross-Species Reactivity Assessment: When working with non-human samples, confirm whether the BTG3 antibody has been validated for your species of interest. Most commercial BTG3 antibodies are optimized for human samples, with predicted homology-based reactivity in other species requiring empirical validation .

• Multi-method Verification: Apply complementary techniques to confirm findings:

  • Perform protein detection using antibodies recognizing different BTG3 epitopes

  • Correlate protein expression with mRNA levels (RT-qPCR or in situ hybridization)

  • Use mass spectrometry to verify pulled-down proteins from immunoprecipitation

• Negative Controls: Include tissue samples where BTG3 expression is known to be absent or minimal, or tissues from BTG3 knockout models if available.

• Absorption Controls: Pre-incubate the antibody with recombinant BTG3 protein or the immunizing peptide prior to staining to confirm that signal elimination occurs when the antibody binding sites are occupied.

• Isotype Controls: Use matched isotype control antibodies at the same concentration to identify non-specific binding.

• Dilution Optimization: Perform careful titration experiments to identify the optimal antibody concentration that maximizes specific signal while minimizing background, particularly for immunohistochemistry applications where tissue autofluorescence can be problematic.

By implementing these rigorous controls, researchers can generate more reliable data when using BTG3 antibodies in complex tissue environments where multiple potential cross-reactive targets may be present.

What are the molecular mechanisms through which BTG3 impairs cell cycle progression from G0/G1 to S phase?

BTG3's role in cell cycle regulation involves several molecular mechanisms that collectively impair G0/G1 to S phase progression:

• Interaction with Cell Cycle Regulators: BTG3 interacts with critical cell cycle machinery, particularly components involved in the G1/S transition. These interactions prevent the formation of active complexes needed for S phase entry.

• CDK Inhibition: Evidence suggests BTG3 can directly or indirectly influence cyclin-dependent kinase (CDK) activity, potentially through:

  • Physical association with CDK/cyclin complexes

  • Modulation of CDK inhibitors (CKIs) like p21 and p27

  • Alteration of CDK phosphorylation status

• E2F Transcription Factor Regulation: BTG3 appears to impact the E2F family of transcription factors, which are crucial for activating genes required for DNA synthesis and S phase entry. This regulation may occur through:

  • Direct protein-protein interactions

  • Influencing upstream regulators like Rb (Retinoblastoma protein)

  • Affecting post-translational modifications of E2F proteins

• Cellular Localization Dynamics: The nuclear-cytoplasmic shuttling of BTG3 may be regulated in a cell cycle-dependent manner, with nuclear localization coinciding with its antiproliferative effects.

To study these mechanisms, researchers employ BTG3 antibodies in various experimental approaches:

Understanding these molecular mechanisms has significant implications for cancer research and developmental biology, given BTG3's expression in the ventricular zone of the developing central nervous system and its potential tumor suppressor functions .

What experimental controls should be included when using BTG3 antibodies in flow cytometry?

Robust flow cytometry experiments with BTG3 antibodies require comprehensive controls to ensure validity and interpretability of results:

• Antibody Validation Controls:

  • Positive Cell Control: Include cell lines with documented BTG3 expression (such as HeLa cells) to confirm antibody performance

  • Negative Cell Control: Use cell lines known to lack BTG3 expression or BTG3 knockout/knockdown cells

  • Antibody Titration: Perform serial dilutions to determine optimal antibody concentration that maximizes signal-to-noise ratio

• Technical Controls:

  • Unstained Control: Cells processed identically but without any antibodies to establish baseline autofluorescence

  • Secondary-Only Control: For indirect staining methods, include samples with only secondary antibody to assess non-specific binding

  • Isotype Control: Include matched isotype antibody at the same concentration to identify non-specific binding due to Fc receptors

  • Fluorescence Minus One (FMO) Control: Include all antibodies in the panel except BTG3 antibody to assess spillover from other fluorochromes

• Biological Controls:

  • Cell Cycle-Specific Controls: Since BTG3 regulates cell cycle progression from G0/G1 to S phase , include samples synchronized at different cell cycle stages to correlate BTG3 expression with cell cycle position

  • Stimulation Controls: If studying conditions that might alter BTG3 expression, include both stimulated and unstimulated samples

• Procedural Controls:

  • Fixation/Permeabilization Controls: Since BTG3 may require intracellular staining, optimize fixation and permeabilization protocols with appropriate controls

  • Viability Dye: Include a viability marker to exclude dead cells, which can bind antibodies non-specifically

• Data Analysis Controls:

  • Compensation Controls: Single-stained controls for each fluorochrome to correct for spectral overlap

  • Gating Strategy Validation: Parallel analysis with alternative methods (e.g., imaging) to confirm subcellular localization

Recommended Control Panel for BTG3 Flow Cytometry:

By implementing this comprehensive control strategy, researchers can generate reliable flow cytometry data using BTG3 antibodies while avoiding common pitfalls in interpretation .

How do post-translational modifications affect BTG3 function and antibody recognition?

Post-translational modifications (PTMs) of BTG3 represent an important yet understudied aspect of its biology that can significantly impact both protein function and antibody recognition:

• Types of PTMs Affecting BTG3:

  • Phosphorylation: Likely regulates BTG3 activity, stability, and interactions with binding partners

  • Ubiquitination: Controls protein turnover and potentially influences subcellular localization

  • SUMOylation: May alter BTG3's interaction with transcription factors and other nuclear proteins

  • Acetylation: Potentially regulates chromatin association and nuclear functions

• Impact on BTG3 Function:

  • Cell Cycle Regulation: PTMs likely modulate BTG3's ability to impair G0/G1 to S phase progression

  • Protein-Protein Interactions: Modifications can create or disrupt binding interfaces

  • Subcellular Localization: PTMs may regulate nuclear-cytoplasmic shuttling

  • Protein Stability: Modifications often determine protein half-life and degradation pathways

• Implications for Antibody Recognition:

  • Epitope Masking: PTMs can physically block antibody access to recognition sites

  • Conformational Changes: Modifications may alter protein folding, affecting discontinuous epitopes

  • Charge Alterations: PTMs like phosphorylation change local charge, potentially disrupting antibody binding

  • Western Blot Migration: Modified BTG3 may show altered migration patterns, appearing as multiple bands

• Experimental Approaches to Address PTM Challenges:

• Research Strategy for PTM-Aware Antibody Selection:

  • Choose antibodies raised against synthetic peptides from regions less likely to undergo PTMs

  • Consider using multiple antibodies targeting different regions of BTG3

  • For PTM-specific studies, use antibodies specifically developed against modified forms of BTG3

  • When studying BRG3 (the E3 ligase), be aware that persulfidation at specific cysteine residues (Cys 206 and Cys 212) significantly impacts its ubiquitination activity

Understanding the interplay between PTMs, BTG3 function, and antibody recognition is crucial for accurate interpretation of experimental results, particularly when studying BTG3's cell cycle regulatory functions in different cellular contexts.

What are the current gaps in understanding BTG3 function, and how might advanced antibody-based techniques help address these?

Despite significant progress in BTG3 research, several important knowledge gaps remain. Advanced antibody-based techniques offer promising approaches to address these outstanding questions:

• Tissue-Specific Functions:

  • Knowledge Gap: While BTG3 is known to be ubiquitously expressed with higher levels in certain tissues (central nervous system, reproductive organs, lung) , the functional significance of this differential expression remains poorly understood.

  • Antibody-Based Approach: Spatial proteomics using highly specific BTG3 antibodies in multiplex immunofluorescence can map expression at cellular resolution across tissues, correlating with functional markers.

• Cell Cycle Regulation Mechanisms:

  • Knowledge Gap: The precise molecular mechanisms by which BTG3 impairs cell cycle progression from G0/G1 to S phase remain incompletely characterized.

  • Antibody-Based Approach: Proximity ligation assays (PLA) with BTG3 antibodies paired with antibodies against cell cycle regulators can identify direct interactions in situ, while ChIP-seq can map genomic binding sites.

• Protein Interaction Networks:

  • Knowledge Gap: The complete interactome of BTG3 across different cellular contexts is not fully mapped.

  • Antibody-Based Approach: BioID or APEX proximity labeling combined with BTG3 antibody-based purification can identify context-specific protein interactions with spatial resolution.

• Post-Translational Regulation:

  • Knowledge Gap: The types, sites, and functional consequences of BTG3 post-translational modifications remain largely uncharacterized.

  • Antibody-Based Approach: Development of modification-specific antibodies, coupled with mass spectrometry validation, can identify regulated modification sites and their functional significance.

• Disease Associations:

  • Knowledge Gap: While BTG3 has potential tumor suppressor functions, its role in specific diseases is not well established.

  • Antibody-Based Approach: Tissue microarray analysis with validated BTG3 antibodies can establish expression patterns across disease states. Single-cell CyTOF with BTG3 antibodies can identify cellular subpopulations with altered expression in disease contexts.

• Developmental Dynamics:

  • Knowledge Gap: Despite high expression in the ventricular zone of the developing central nervous system , BTG3's role in neurodevelopment remains poorly characterized.

  • Antibody-Based Approach: Temporal immunohistochemistry studies with BTG3 antibodies during development, combined with lineage markers, can clarify developmental functions.

Research Priority Matrix for BTG3 Knowledge Gaps:

By strategically applying these advanced antibody-based approaches, researchers can address fundamental questions about BTG3 biology and potentially uncover new therapeutic targets related to its cell cycle regulatory functions.

How can researchers assess interactions between BTG3 and other proteins using immunoprecipitation followed by mass spectrometry?

Immunoprecipitation followed by mass spectrometry (IP-MS) represents a powerful approach for identifying and characterizing BTG3 protein interactions. This methodology requires careful experimental design and execution:

• Experimental Design Considerations:

  • Cell/Tissue Selection: Choose models with verified BTG3 expression; HeLa cells are documented to express BTG3 and serve as a good starting point

  • Experimental Conditions: Consider both basal and stimulated conditions that might regulate BTG3 interactions

  • Controls: Include IgG control IPs and, ideally, BTG3 knockout/knockdown samples

  • Crosslinking Options: Evaluate whether crosslinking is needed to capture transient interactions

• BTG3 Antibody Selection Criteria:

  • Epitope Location: Select antibodies targeting regions unlikely to interfere with protein-protein interactions

  • Validation Status: Use antibodies validated for immunoprecipitation applications

  • Format Considerations: Consider whether native or denatured IP conditions are optimal

  • Species Compatibility: Ensure compatibility with downstream MS detection systems

• Optimized IP-MS Protocol Framework:

  • Cell Lysis: Use gentle lysis conditions to preserve protein complexes

  • Pre-clearing: Remove non-specific binding proteins with control beads

  • Immunocapture: Incubate lysates with BTG3 antibody pre-bound to beads

  • Washing: Perform stringent washes while preserving specific interactions

  • Elution: Choose between native elution (competition) or denaturing conditions

  • MS Analysis: Implement appropriate MS/MS methods for protein identification

• Data Analysis Strategy:

  • Filtering Criteria: Compare to IgG control and exclude common contaminants

  • Quantification Approach: Use label-free or labeling methods (TMT, SILAC) for quantification

  • Interaction Confidence: Assign confidence scores based on peptide counts, specificity, and reproducibility

  • Network Analysis: Map interactions to known pathways and protein complexes

• Validation of Identified Interactions:

  • Reciprocal IP: Confirm key interactions by IP with antibodies against identified partners

  • Proximity Ligation Assay: Visualize interactions in situ

  • Functional Studies: Test biological relevance through knockdown or mutation of interaction interfaces

Sample Workflow for BTG3 IP-MS:

This approach allows researchers to move beyond candidate-based interaction studies to unbiased discovery of the BTG3 interactome, particularly important for understanding BTG3's role in cell cycle regulation from G0/G1 to S phase .

What approaches can be used to study the role of BTG3 in different tissue contexts, given its ubiquitous expression pattern?

BTG3's ubiquitous expression with tissue-specific enrichment presents both challenges and opportunities for contextual functional studies. Researchers can employ several sophisticated approaches to elucidate tissue-specific roles:

• Spatial Expression Mapping Strategies:

  • Multiplexed Immunohistochemistry: Use BTG3 antibodies in conjunction with tissue-specific markers to identify cell populations with high expression

  • Single-Cell Protein Analysis: Apply techniques like CITE-seq or CyTOF with BTG3 antibodies to correlate expression with cell identity

  • In Situ Hybridization + Immunofluorescence: Combine BTG3 mRNA detection with protein localization to assess transcriptional vs. post-transcriptional regulation

  • Spatial Transcriptomics: Correlate BTG3 expression with tissue architecture and microenvironment

• Tissue-Specific Functional Assessment:

  • Conditional Knockout Models: Generate tissue-specific BTG3 deletion models focusing on high-expression tissues (ventricular zone of CNS, reproductive tissues, thymus, lung)

  • Ex Vivo Tissue Cultures: Manipulate BTG3 expression in tissue explants to assess acute functional consequences

  • Organoid Systems: Derive organoids from tissues with high BTG3 expression to study function in 3D culture

  • Patient-Derived Xenografts: Examine BTG3 function in humanized models maintaining tissue architecture

• Protein Interaction Network Analysis:

  • Tissue-Specific Interactome Mapping: Perform BTG3 immunoprecipitation followed by mass spectrometry across different tissues

  • Cell Type-Resolved Proximity Labeling: Use tissue-specific promoters to drive expression of BTG3-BioID fusions

  • Comparative Network Analysis: Identify tissue-specific vs. universal BTG3 interaction partners

• Functional Readouts in Tissue Context:

  • Cell Cycle Analysis in Tissue Sections: Combine BTG3 antibody staining with proliferation markers (Ki67, BrdU) to assess correlation with cell cycle across tissues

  • Lineage Tracing with BTG3 Modulation: Track developmental consequences of BTG3 manipulation in tissues with high expression

  • Primary Cell Isolation and Culture: Compare BTG3 function in primary cells derived from different tissues

Comparative Analysis Framework for Tissue-Specific BTG3 Studies:

• Technological Approaches for Tissue-Specific Studies:

  • Antibody-Based Tissue Cytometry: Quantitative assessment of BTG3 levels across tissue sections

  • In Vivo CRISPR Screens: Tissue-specific delivery of BTG3-targeting guides to assess function

  • Spatial Multi-omics: Integrate proteomic, transcriptomic and epigenomic data with spatial resolution

By systematically applying these approaches across tissues with differential BTG3 expression, researchers can deconvolute the general cell cycle regulatory functions of BTG3 from tissue-specific roles that may involve unique protein interactions or regulatory mechanisms.