BTG4 Antibody

What is BTG4 and why is it significant in developmental biology?

BTG4 is an adapter protein belonging to the BTG/Tob family involved in cell growth regulation. It functions by bridging CNOT7, a catalytic subunit of the CCR4-NOT complex, to EIF4E. This interaction facilitates maternal mRNA decay during oocyte maturation and in fertilized eggs, making it essential for the maternal-zygotic transition (MZT), zygotic cleavage, and initiation of embryonic development . The significance of BTG4 in developmental biology stems from its crucial role in preventing spontaneous activation of oocytes and maintaining metaphase II arrest, which is vital for successful fertilization and early embryonic development .

What are the basic characteristics of commercially available BTG4 antibodies?

Commercial BTG4 antibodies are available in both polyclonal and monoclonal formats, primarily derived from rabbit hosts. These antibodies are designed to target specific regions of the BTG4 protein and can be used across multiple applications. The most common BTG4 antibodies have the following specifications:

The observed molecular weight (28-30 kDa) often differs slightly from the calculated molecular weight (26 kDa), which is important to note when evaluating western blot results .

How should I select the appropriate BTG4 antibody for my specific research application?

When selecting a BTG4 antibody, consider these key factors based on your experimental needs:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IF, etc.). For example, some antibodies perform well in Western blot (1:500-1:1000 dilution) but may require different concentrations for IHC (1:20-1:200) .

• Species reactivity: Confirm that the antibody recognizes BTG4 in your experimental species. The search results indicate antibodies that react with human, mouse, and rat samples .

• Epitope specificity: Choose antibodies that target regions conserved in your species of interest. Some antibodies target recombinant fragments within human BTG4 from amino acid 1 to the C-terminus .

• Validation evidence: Look for antibodies with published validation data, especially those cited in peer-reviewed publications. This provides confidence in antibody performance for specific experimental conditions .

• Clonality considerations: For detecting specific isoforms or when high reproducibility is needed, monoclonal antibodies like EPRZJU-21 may be preferable. For broader detection or when signal amplification is desired, polyclonal antibodies may be more suitable .

Select antibodies that have been validated in tissues where BTG4 is known to be expressed, such as testicular, ovarian, and brain tissues .

What are the optimal protocols for using BTG4 antibodies in Western blot applications?

For optimal Western blot results with BTG4 antibodies, follow these methodological guidelines:

• Sample preparation: Use fresh tissue lysates from relevant sources such as ovary, testis, or brain tissue. For cell lines, consider using BTG4-transfected cells as positive controls since endogenous expression may be limited in many cell lines .

• Protein loading: Load 20 μg of total protein per lane for adequate detection. This amount has been validated in published protocols for detecting BTG4 in various samples .

• Blocking conditions: Use 5% non-fat dry milk (NFDM) in TBST as the blocking/dilution buffer to minimize background and optimize specific signal .

• Antibody dilution: For polyclonal antibodies, use at 1:500-1:1000 dilution; for monoclonal antibodies like EPRZJU-21, a 1:1000 dilution is recommended .

• Detection system: Use appropriate secondary antibodies, such as goat polyclonal to rabbit IgG at 1:50000 dilution for rabbit primary antibodies .

• Expected results: Anticipate a band at approximately 26-30 kDa. Note that the observed molecular weight (28-30 kDa) may differ from the predicted size (26 kDa) .

• Controls: Include appropriate positive controls (e.g., mouse brain lysate, human ovary lysate) and negative controls (e.g., lysates from cells transfected with empty vectors) .

For troubleshooting purposes, consider that detection may be challenging in samples with low endogenous BTG4 expression. In such cases, enrichment through immunoprecipitation before Western blot analysis may improve results.

What are the recommended protocols for immunohistochemical detection of BTG4?

For successful immunohistochemical detection of BTG4 in tissue sections, implement the following protocol:

• Tissue preparation: Use paraffin-embedded tissue sections from relevant tissues such as ovary, testis, or brain. Ensure proper fixation to preserve antigen integrity .

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal antigen retrieval. Alternatively, citrate buffer at pH 6.0 can be used, although this may affect sensitivity .

• Blocking: Block endogenous peroxidase activity and non-specific binding sites using appropriate blocking reagents based on your detection system.

• Antibody dilution: For IHC applications, use antibodies at 1:20-1:200 dilution, with specific optimization required for each tissue type and antibody .

• Incubation conditions: Incubate with primary antibody overnight at 4°C for optimal binding and specificity.

• Detection system: Use an appropriate detection system compatible with your primary antibody (e.g., HRP-conjugated secondary antibody with DAB substrate).

• Counterstaining: Lightly counterstain with hematoxylin to visualize tissue morphology while maintaining clear visualization of the specific BTG4 signal.

• Controls: Include positive control tissues (human ovarian or testicular tissue) where BTG4 is known to be expressed, as well as negative controls (omitting primary antibody) .

The protocol should be optimized for each specific tissue and experimental condition, as BTG4 expression levels vary significantly between tissues and developmental stages.

How can I validate the specificity of BTG4 antibodies in my experimental system?

To ensure antibody specificity and validate results in your BTG4 research, implement these comprehensive validation approaches:

• Genetic validation: Use tissue or cells from BTG4 knockout/knockdown models as negative controls. Compare these with wild-type samples to confirm specificity of the detected signal .

• Recombinant protein controls: Use cells transfected with BTG4 expression vectors as positive controls. Compare with cells transfected with empty vectors to confirm specificity .

• Peptide competition assay: Pre-incubate the antibody with excess BTG4 immunogenic peptide before application to samples. Specific signals should be significantly reduced or eliminated.

• Cross-reactivity assessment: Test the antibody against closely related family members (other BTG family proteins) to ensure it doesn't cross-react with these proteins.

• Multiple antibody validation: Use more than one antibody targeting different epitopes of BTG4 to confirm consistent detection patterns.

• Multiple detection methods: Validate findings using complementary techniques such as Western blot, IHC, and immunofluorescence to confirm consistent localization and expression patterns.

• Correlation with mRNA expression: Perform qRT-PCR analysis to correlate protein detection with mRNA expression levels in the same samples .

• Size verification: Confirm that the detected protein band appears at the expected molecular weight (26-30 kDa) in Western blot applications .

This multi-faceted validation approach ensures that observed signals truly represent BTG4 expression and localization, increasing confidence in experimental results.

How can BTG4 antibodies be used to investigate maternal-zygotic transition mechanisms?

BTG4 antibodies provide powerful tools for investigating the maternal-zygotic transition (MZT) through these methodological approaches:

• Temporal expression analysis: Use BTG4 antibodies in Western blot and immunofluorescence to track BTG4 protein levels and localization throughout oocyte maturation, fertilization, and early embryonic development. This reveals when BTG4 is most active during MZT .

• Co-immunoprecipitation studies: Utilize BTG4 antibodies for co-IP experiments to identify and confirm its interactions with other proteins involved in MZT, particularly components of the CCR4-NOT deadenylase complex such as CNOT7 and CNOT8, as well as poly(A)-binding proteins like PABPN1 .

• Immunofluorescence co-localization: Combine BTG4 antibodies with antibodies against proteins involved in mRNA decay pathways to visualize their spatial relationships during different stages of oocyte maturation and early development.

• Chromatin immunoprecipitation (ChIP): Apply BTG4 antibodies in ChIP assays to investigate potential direct or indirect interactions with chromatin during the transition from maternal to zygotic gene expression.

• Proximity ligation assays: Use BTG4 antibodies in conjunction with antibodies against interaction partners like CNOT7, CNOT8, or EIF4E to visualize and quantify protein-protein interactions in situ during MZT .

• Correlative microscopy: Combine immunofluorescence using BTG4 antibodies with RNA visualization techniques to correlate BTG4 localization with sites of active mRNA decay during MZT.

These approaches can reveal how BTG4 orchestrates the large-scale deadenylation and degradation of maternal mRNAs at the MI-MII transition, which is crucial for proper embryonic development .

What are the challenges in detecting endogenous BTG4 in different cell and tissue types?

Detecting endogenous BTG4 presents several challenges that researchers should anticipate and address:

• Tissue-specific expression patterns: BTG4 is predominantly expressed in reproductive tissues, particularly in testicular and ovarian tissues, making detection in other tissues challenging due to low expression levels . This necessitates optimization of detection protocols for each tissue type.

• Temporal expression dynamics: BTG4 expression varies significantly during oocyte maturation and early embryonic development, with synthesis reported to occur specifically during metaphase II. This temporal specificity requires careful timing of sample collection .

• Post-translational modifications: Potential post-translational modifications can affect antibody recognition and cause variations in apparent molecular weight (observed at 28-30 kDa versus predicted 26 kDa) .

• Cross-reactivity with BTG family members: The BTG family shares structural similarities, potentially leading to cross-reactivity. Thorough validation is necessary to ensure specificity for BTG4 rather than other family members.

• Subcellular localization challenges: BTG4 may shuttle between different subcellular compartments, requiring careful fixation and permeabilization protocols for accurate localization studies.

• Antibody sensitivity limitations: Low endogenous expression levels may fall below detection thresholds of some antibodies, necessitating signal amplification techniques or concentrating steps like immunoprecipitation before analysis.

• Species-specific variations: Sequence differences between species can affect antibody recognition, requiring validation for each species of interest .

To overcome these challenges, consider using tissues with known high BTG4 expression as positive controls, employing signal amplification techniques for low-expression samples, and validating results with multiple detection methods and antibodies targeting different epitopes.

How can BTG4 antibodies be used to investigate infertility and embryonic development disorders?

BTG4 antibodies can be powerful tools for investigating infertility and embryonic development disorders through these methodological approaches:

• Comparative expression analysis: Compare BTG4 protein levels and localization in oocytes and embryos from fertile versus infertile subjects using quantitative immunofluorescence or Western blot analysis. Aberrant expression patterns may correlate with specific fertility issues .

• Mutation-specific antibodies: Develop or use antibodies that specifically recognize wild-type BTG4 versus mutant forms identified in infertility cases, such as those with homozygous mutations causing zygotic cleavage failure .

• Functional domain studies: Use antibodies targeting specific functional domains of BTG4 (like the W95A mutation site that abolishes interaction with CNOT7/8) to assess how domain-specific mutations affect protein-protein interactions in patient samples .

• Immunohistochemical screening: Apply BTG4 antibodies in IHC screening of ovarian or testicular biopsies from infertility patients to identify abnormal expression or localization patterns as potential diagnostic markers .

• Therapuetic target assessment: Use BTG4 antibodies to monitor the effects of experimental treatments aimed at correcting BTG4-related defects in infertility models.

• Genetic correlation studies: Combine BTG4 immunostaining with genetic analysis to correlate specific BTG4 mutations with protein expression and localization patterns in patient samples .

• APC/C activity correlation: Use BTG4 antibodies alongside markers of anaphase-promoting complex/cyclosome (APC/C) activity to investigate how BTG4 deficiencies affect cell cycle regulation in oocytes and embryos .

This research is particularly relevant given that homozygous mutations in BTG4 have been directly linked to zygotic cleavage failure, and BTG4 is required for inhibiting the anaphase-promoting complex/cyclosome during metaphase II arrest, a critical step for successful fertilization .

What are common issues in BTG4 Western blot experiments and how can they be resolved?

Researchers frequently encounter these challenges when performing Western blot for BTG4 detection:

When working with reproductive tissues, remember that BTG4 expression is dynamic and stage-specific, requiring precise timing of sample collection for reproducible results. For challenging samples, consider using immunoprecipitation to concentrate BTG4 before Western blot analysis.

How can I distinguish between specific and non-specific signals in BTG4 immunostaining experiments?

To differentiate between specific and non-specific signals in BTG4 immunostaining, implement these methodological controls and analytical approaches:

• Negative controls: Always include technical negative controls by omitting the primary antibody while maintaining all other staining steps. Additionally, use biological negative controls such as tissues known not to express BTG4 or samples where BTG4 has been knocked down/out .

• Absorption controls: Pre-incubate the BTG4 antibody with excess immunizing peptide before application to serial sections of the same sample. Specific staining should be abolished or significantly reduced, while non-specific signals will remain.

• Correlation with known expression patterns: Compare your staining pattern with well-established expression profiles of BTG4. For instance, strong expression should be detectable in testicular and ovarian tissues, particularly in oocytes during specific developmental stages .

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of BTG4. Specific signals should show consistent patterns across different antibodies, while non-specific signals typically vary.

• Subcellular localization analysis: Evaluate whether the observed staining pattern matches the expected subcellular localization of BTG4. Discrepancies may indicate non-specific binding.

• Signal intensity gradient: Perform serial dilutions of the primary antibody. Specific signals should decrease proportionally with antibody dilution, while non-specific background often remains relatively constant.

• Comparison with mRNA expression: Correlate immunostaining results with mRNA expression data from in situ hybridization or qRT-PCR from the same tissues or developmental stages .

For immunohistochemistry specifically, use appropriate antigen retrieval methods (TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0) to enhance specific signal while minimizing background .

How should I interpret contradictory results between different BTG4 antibodies?

When faced with contradictory results between different BTG4 antibodies, implement this systematic approach to resolve discrepancies:

• Epitope mapping analysis: Compare the epitope regions targeted by each antibody. Discrepancies may arise when antibodies target different domains of BTG4, especially if some domains are masked by protein-protein interactions or post-translational modifications in certain contexts .

• Antibody validation comparison: Evaluate the validation evidence for each antibody, including published data, vendor validation, and your own controls. Prioritize results from antibodies with more extensive validation .

• Clonality consideration: Analyze whether discrepancies occur between monoclonal and polyclonal antibodies. Monoclonal antibodies (like EPRZJU-21) offer higher specificity but may miss some protein conformations, while polyclonal antibodies provide broader detection but potentially higher background .

• Application-specific optimization: Determine if contradictions are application-specific. Some antibodies perform well in Western blot but poorly in IHC or vice versa, requiring application-specific optimization .

• Experimental condition analysis: Assess whether discrepancies result from differences in experimental conditions rather than antibody performance. Standardize conditions across experiments with different antibodies.

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other BTG family members, especially in tissues where multiple family members are expressed.

• Functional validation approaches: Move beyond detection methods to functional assays. For example, if one antibody detects a putative BTG4-CNOT7 interaction while another doesn't, validate using co-immunoprecipitation or proximity ligation assays .

• Genetic models: When possible, use genetic models (knockout/knockdown) to definitively determine which antibody accurately reflects BTG4 expression patterns .

When reporting contradictory results, clearly document the specific antibodies used (including catalog numbers), experimental conditions, and potential limitations to advance the field's understanding of BTG4 detection challenges.

How are BTG4 antibodies being used in cutting-edge reproductive biology research?

BTG4 antibodies are enabling several innovative research directions in reproductive biology:

• Single-cell protein profiling: Researchers are using BTG4 antibodies in single-cell Western blot and mass cytometry approaches to investigate cell-to-cell variability in BTG4 expression within oocyte populations, potentially explaining differential developmental competence .

• Super-resolution microscopy applications: BTG4 antibodies combined with super-resolution microscopy techniques are revealing previously undetectable subcellular localization patterns and dynamic changes during oocyte maturation and fertilization .

• Functional domain mapping: New antibodies targeting specific functional domains are helping characterize how the adapter function of BTG4 (bridging CNOT7 to EIF4E) mechanistically controls maternal mRNA decay .

• Clinical diagnostics development: BTG4 antibodies are being evaluated as diagnostic tools to assess oocyte quality and predict developmental potential in assisted reproductive technologies, based on the protein's critical role in maternal-zygotic transition .

• Evolutionary conservation studies: Comparative analyses using BTG4 antibodies across species (from mice to humans and other mammals) are revealing evolutionary conservation and divergence in mechanisms controlling early embryonic development .

• Therapeutic target validation: As understanding of BTG4's role in fertility grows, antibodies are helping validate it as a potential therapeutic target for certain forms of infertility, particularly those involving defects in oocyte maturation or early embryonic development .

• Proteome-wide interaction mapping: Advanced proteomics approaches using BTG4 antibodies for immunoprecipitation coupled with mass spectrometry are expanding our understanding of BTG4's interactome beyond known partners like CNOT7/8, PABPN1, and EIF4E .

These cutting-edge applications are significantly advancing our understanding of the molecular mechanisms controlling the oocyte-to-embryo transition and opening new possibilities for addressing infertility issues.

What emerging technologies are enhancing the specificity and sensitivity of BTG4 detection?

Several emerging technologies are revolutionizing BTG4 detection with enhanced specificity and sensitivity:

• Proximity ligation assays (PLA): This technology allows visualization of protein-protein interactions in situ with high specificity by detecting proteins that are in close proximity (<40 nm). It's being applied to study BTG4 interactions with CNOT7/8 and other partners in their native cellular context .

• Single-molecule imaging: Techniques like single-molecule pull-down and coincidence detection are beginning to reveal the stoichiometry and dynamics of BTG4 interactions with deadenylase complexes during mRNA decay.

• CRISPR epitope tagging: Endogenous tagging of BTG4 using CRISPR/Cas9 technology facilitates detection with highly specific anti-tag antibodies, circumventing issues with direct BTG4 antibody specificity while preserving native expression patterns and levels.

• Nanobodies and recombinant antibody fragments: These smaller binding molecules offer improved tissue penetration and access to epitopes that may be obscured from conventional antibodies, particularly useful for studying BTG4 in complex with its binding partners .

• Mass cytometry (CyTOF): Metal-conjugated antibodies against BTG4 and related proteins enable highly multiplexed protein detection without spectral overlap issues, allowing comprehensive analysis of BTG4 in relation to dozens of other proteins simultaneously.

• Imaging mass cytometry: This technique combines the high-parameter capabilities of mass cytometry with high-resolution imaging, enabling spatial analysis of BTG4 expression in tissue contexts with unprecedented multiplexing capabilities.

• Microfluidic immunofluorescence: Automated microfluidic platforms improve reproducibility and sensitivity of BTG4 detection while using minimal sample volumes, crucial for precious samples like oocytes and early embryos.

• Aptamer-based detection: DNA/RNA aptamers selected for high-affinity binding to BTG4 offer alternatives to traditional antibodies with potentially improved specificity and reduced batch-to-batch variation.

These technological advances are overcoming traditional limitations in BTG4 detection, enabling more detailed studies of its expression, localization, and function in reproductive biology and embryonic development.

How might understanding BTG4 function contribute to advancements in assisted reproductive technologies?

Understanding BTG4 function through antibody-based research could transform assisted reproductive technologies (ARTs) in several significant ways:

• Improved oocyte quality assessment: BTG4 immunostaining patterns could serve as biomarkers for oocyte developmental competence, potentially replacing morphological assessment with molecular profiling. Abnormal BTG4 expression or localization might identify oocytes with compromised developmental potential before fertilization .

• Personalized fertility treatment approaches: Screening patients' oocytes for BTG4 expression and function could guide personalized treatment strategies, particularly for patients with unexplained infertility or repeated IVF failure .

• Optimized oocyte cryopreservation: Understanding how cryopreservation affects BTG4 stability and function could lead to improved protocols that preserve this critical protein's activity, enhancing post-thaw developmental competence.

• Novel therapeutic interventions: Detailed knowledge of how BTG4 regulates maternal mRNA degradation could inspire mRNA-based therapeutics to temporarily supplement or modulate BTG4 function in compromised oocytes .

• Genetic screening enhancements: Screening embryos for BTG4 mutations could identify those at risk for developmental failure, as homozygous mutations in BTG4 have been linked to zygotic cleavage failure .

• Culture media optimization: Understanding BTG4's role in maternal-zygotic transition could inform the development of stage-specific culture media compositions that support optimal BTG4 function during critical developmental transitions .

• Improved in vitro maturation protocols: Knowledge of BTG4's role during oocyte maturation could enhance in vitro maturation protocols for immature oocytes, potentially expanding the pool of viable oocytes for patients with limited ovarian reserve.

• Male fertility applications: Given BTG4's expression in testicular tissue, understanding its role in spermatogenesis might also lead to new approaches for addressing certain forms of male infertility .

These advancements could significantly improve success rates in assisted reproduction while reducing the emotional and financial burden of repeated unsuccessful cycles, particularly for patients with currently unexplained fertility challenges related to early embryonic development failure.