ESRRB Antibody, Biotin conjugated

What is ESRRB and what is its molecular structure?

ESRRB (Estrogen-Related Receptor beta) is a nuclear receptor belonging to the NR3 Steroid Receptor family with a calculated molecular weight of approximately 56 kDa (508 amino acids), though observed molecular weight in experiments typically ranges between 48-56 kDa . The protein functions in transcriptional regulation, early placental development, and acts as a repressor of glucocorticoid receptor-mediated transcriptional activity . ESRRB can bind as a monomer to extended half-site sequences (TNAAGTGCA) or as a homodimer to estrogen response elements (ERE), palindromic thyroid hormone response elements (TRE(pal)), and SF-1 response elements .

What are the primary applications for biotin-conjugated ESRRB antibodies?

Biotin-conjugated antibodies are particularly valuable in research applications requiring high sensitivity and versatility. Based on similar estrogen receptor antibodies, biotin-conjugated ESRRB antibodies are suitable for:

While standard ESRRB antibodies are validated for Western Blot (WB), Immunoprecipitation (IP), Immunofluorescence (IF), and Immunohistochemistry (IHC) , the biotin conjugation enhances detection sensitivity and provides additional experimental flexibility.

How should samples be prepared for immunohistochemistry using biotin-conjugated ESRRB antibodies?

For optimal results in formalin-fixed, paraffin-embedded (FFPE) tissues:

• Deparaffinize and rehydrate tissue sections following standard protocols

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Block endogenous peroxidase activity with 0.3% H₂O₂ for 10 minutes

• Critical step: Block endogenous biotin using a commercial biotin-blocking kit to prevent non-specific binding

• Block non-specific binding with 1% BSA in PBST for 1 hour

• Apply optimally diluted biotin-conjugated ESRRB antibody and incubate overnight at 4°C

• Wash extensively with PBST

• Detection can be performed with streptavidin-HRP followed by chromogenic substrate

This protocol is adapted from established methods for estrogen receptor antibody applications in immunohistochemistry .

How can biotinylated ESRRB antibodies be utilized in proximity labeling experiments?

Biotinylated antibodies are particularly valuable for proximity labeling approaches such as BAR (Biotinylation by Antibody Recognition). This technique allows researchers to investigate the proximal proteome of ESRRB in its native cellular context:

• Fix cells with 1% formaldehyde to preserve protein-protein interactions

• Permeabilize cells with 0.1% Triton X-100

• Block with 1% BSA in PBST for 1 hour

• Incubate with biotin-conjugated ESRRB antibody (or primary antibody followed by HRP-conjugated secondary antibody)

• Add labeling buffer containing 140 μM biotin phenol and 0.03% H₂O₂ in PBST for 3 minutes

• Quench the reaction with 500 mM sodium ascorbate

• Lyse cells and capture biotinylated proteins using streptavidin beads

• Analyze by mass spectrometry to identify proteins in close proximity to ESRRB

This approach offers significant advantages over traditional immunoprecipitation by capturing transient and weak interactions in their spatial context, without requiring genetic modification of target cells .

What is the relationship between ESRRB and classical estrogen receptors (ERα)?

While ESRRB and ERα (ESR1) are both members of the nuclear receptor family, they exhibit distinct functional and regulatory properties:

• Binding differences: ESRRB can bind as a monomer to extended half-site sequences and as a homodimer to estrogen response elements (ERE) , while ERα primarily functions through ERE-dependent and ERE-independent signaling pathways .

• Functional interactions: ERα is involved in ligand-dependent nuclear transactivation through direct DNA binding or association with other transcription factors like AP-1/c-Jun, c-Fos, and Sp1 . In contrast, ESRRB is primarily associated with pluripotency maintenance in embryonic stem cells and early development processes.

• Proximal proteome differences: Studies using proximity labeling techniques reveal distinct protein interaction networks between these receptors. ERα interacts with various coregulatory proteins implicated in breast cancer progression and drug resistance , while ESRRB forms regulatory networks with pluripotency factors.

Understanding these distinctions is crucial when designing experiments targeting specific estrogen-related receptor pathways.

How does ESRRB function in transcriptional regulatory networks?

ESRRB participates in complex transcriptional regulatory networks, particularly in embryonic stem cells:

• Forms a regulatory loop with Dax1 and Oct3/4 for controlling pluripotency and self-renewal in embryonic stem cells

• Binds to specific Estrogen-Related Receptor Elements (ERREs) within target genes

• Multiple binding sites have been identified in the Esrrb gene itself (ERRE1, ERRE2, ERRE3, and ERRE4), suggesting autoregulation

• Functions both as an activator and repressor of transcription depending on cellular context and cofactor recruitment

Researchers investigating ESRRB regulatory networks should consider performing ChIP-seq experiments using biotin-conjugated ESRRB antibodies to map genomic binding sites comprehensively.

What validation steps should be performed for biotin-conjugated ESRRB antibodies?

Proper validation is essential before using biotin-conjugated ESRRB antibodies in critical experiments:

• Specificity testing: Perform Western blot analysis in positive control samples (e.g., PC-13 cells, A549 cells for human ESRRB) alongside negative controls (ESRRB knockout/knockdown cells)

• Signal verification: For immunohistochemistry applications, include both positive controls (tissues known to express ESRRB) and negative controls (primary antibody omission, isotype controls)

• Biotinylation confirmation: Verify successful biotinylation using streptavidin-HRP detection systems or immunofluorescence with fluorescently-labeled streptavidin

• Functionality assessment: Test the antibody in its intended application at different dilutions to establish optimal working conditions

• Cross-reactivity evaluation: If working across species, validate species reactivity as reported ranges include human, mouse, and rat for many ESRRB antibodies

What are the recommended dilutions and experimental conditions for different applications?

Based on established protocols for ESRRB antibodies, the following guidelines can be adapted for biotin-conjugated versions:

These recommendations should be optimized for each specific biotin-conjugated ESRRB antibody and experimental system.

How can non-specific binding be minimized when using biotin-conjugated antibodies?

Biotin-conjugated antibodies face unique challenges regarding non-specific binding due to endogenous biotin in many biological samples:

• Endogenous biotin blocking: Critical pre-treatment with commercial avidin/biotin blocking kits before antibody application

• Optimized blocking solution: Use 1% BSA in PBST for 1 hour at room temperature , considering alternative blockers (5% normal serum, commercial blockers) if background persists

• Stringent washing: Implement extended washing steps (at least 1 hour with PBST for proximity labeling protocols)

• Antibody dilution optimization: Titrate to determine minimum effective concentration that maintains specific signal while reducing background

• Secondary detection optimization: When using streptavidin conjugates, include appropriate controls to distinguish between specific binding and endogenous biotin detection

How can weak or absent signals be addressed when using biotin-conjugated ESRRB antibodies?

When encountering weak or absent signals:

• Epitope accessibility: ESRRB functions primarily as a nuclear receptor; ensure nuclear permeabilization is complete for intracellular applications

• Expression verification: Confirm ESRRB expression in your samples through RT-PCR or with alternative antibodies, as expression levels may vary across cell types and developmental stages

• Antigen retrieval optimization: For FFPE tissues, test multiple antigen retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Detection sensitivity: Utilize signal amplification systems (TSA, ABC) for low-abundance targets

• Sample preparation: Ensure proper sample preparation preserves the ESRRB epitope (avoid excessive heat or harsh detergents)

How should results from proximity labeling experiments using biotin-conjugated ESRRB antibodies be validated?

Proximity labeling results require rigorous validation:

• Control experiments: Include IgG control antibodies and cells lacking ESRRB expression

• Orthogonal validation: Confirm key protein interactions using co-immunoprecipitation, PLA (Proximity Ligation Assay), or FRET approaches

• Functional validation: Perform knockdown/knockout studies of identified interacting partners to assess functional relevance

• Biotinylation confirmation: Verify biotinylation success through immunofluorescence on aliquot samples prior to mass spectrometry analysis

• Statistical analysis: Apply appropriate statistical methods to distinguish true ESRRB-proximal proteins from background

This validation strategy follows established approaches used in ERα proximal proteome studies, which can be adapted for ESRRB research .

How can biotin-conjugated ESRRB antibodies advance studies of pluripotency and stem cell biology?

ESRRB plays critical roles in pluripotency networks, and biotin-conjugated antibodies offer unique research opportunities:

• Dynamic interaction mapping: Using BAR technology adapted for ESRRB, researchers can map temporal changes in protein interactions during differentiation or reprogramming processes

• Chromatin landscape analysis: Combining biotin-conjugated ESRRB antibodies with proximity labeling can reveal chromatin modifiers and transcriptional machinery associated with ESRRB binding sites

• Single-cell applications: New technologies allowing proximity labeling in single cells could reveal heterogeneity in ESRRB regulatory networks within stem cell populations

• In vivo applications: BAR technology has been successfully applied to primary human tissues , suggesting potential for analyzing ESRRB interactions in complex in vivo environments

What are the considerations for using biotin-conjugated ESRRB antibodies in cancer research?

As nuclear receptors are increasingly recognized as therapeutic targets in cancer, ESRRB research holds promise:

• Resistance mechanisms: Similar to ERα studies, ESRRB proximity proteomics could reveal adaptation mechanisms to targeted therapies

• Biomarker development: Spatial analysis of ESRRB interactions might identify novel biomarkers for cancer prognosis or treatment response

• Methodological advantages: The BAR approach using biotin-conjugated antibodies avoids the need for genetic modification of patient-derived samples, making it suitable for translational cancer research

• Therapeutic targeting: Identifying critical ESRRB interactions could reveal new therapeutic vulnerabilities in cancers expressing this nuclear receptor