ESRRB Antibody, HRP conjugated

What is ESRRB and what is its biological significance?

ESRRB (Estrogen-related receptor beta) is a transcription factor belonging to the orphan nuclear receptor family. It functions by binding to a canonical ESRRB recognition sequence (ERRE) 5'TCAAGGTCA-3' located on promoters and enhancers of target genes, thereby regulating their expression or transcriptional activity. ESRRB plays crucial roles in maintaining self-renewal and pluripotency of embryonic and trophoblast stem cells through various signaling pathways, including FGF and Wnt signaling .

Within stem cell biology, ESRRB has been shown to block rapid differentiation of trophoblast stem cells (TSCs) in the absence of Fgf4 and enable accelerated proliferation rates. It functions by upregulating self-renewal markers including Cdx2, Eomes, and Elf5, while inhibiting expression of differentiation markers such as Gcm1, Mash2, and Tpbpa . Recent studies have also identified ESRRB as a cell cycle-dependent XEN (extra-embryonic endoderm) priming factor that is upregulated during the G2/M phase .

What are the specifications of the ESRRB Antibody, HRP conjugated?

The ESRRB Antibody, HRP conjugated (Product Code: CSB-PA007836LB01HU) has the following specifications:

The antibody recognizes the human steroid hormone receptor ERR2 (ERR beta-2), also known as Estrogen-related receptor beta (ERR-beta) or Nuclear receptor subfamily 3 group B member 2 .

How should ESRRB Antibody, HRP conjugated be stored and handled for optimal performance?

For optimal performance and longevity of the ESRRB Antibody, HRP conjugated:

• Upon receipt, store at either -20°C or -80°C

• Avoid repeated freeze-thaw cycles, as this can denature and fragment the antibody

• The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• When handling, use sterile techniques and appropriate personal protective equipment

• For short-term use (within 1-2 weeks), the antibody may be stored at 4°C

• Aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles

• When thawing, allow the antibody to reach room temperature naturally before use

• Centrifuge the vial briefly before opening to ensure all solution is at the bottom of the tube

How can ESRRB Antibody, HRP conjugated be used in ChIP-seq studies to identify ESRRB binding sites?

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with ESRRB antibodies has provided significant insights into ESRRB's genomic targets. When designing ChIP-seq experiments using the ESRRB Antibody, HRP conjugated, researchers should consider:

• Cross-linking optimization: Although HRP-conjugated antibodies are not typically preferred for ChIP-seq, the underlying ESRRB antibody can be used with standard cross-linking conditions (1% formaldehyde for 10 minutes).

• Target enrichment: Research has shown that ESRRB peaks are predominantly enriched at promoters and enhancers. ChIP-seq analysis in trophoblast stem cells has revealed significant overlap between different ESRRB target datasets .

• Motif identification: Following ESRRB ChIP-seq, motif analysis using MEME/DREME followed by Tomtom has identified that ESRRB peaks are highly enriched in TSC-specific genes. Additionally, Esrrb/Esrra-binding motifs have been identified, suggesting potential self-reinforcing functions .

• Enhancer identification: ESRRB has been found to associate with enhancers of genes like Gata6, Gata4, Foxa2, Dab2, and Foxq1. These enhancers are typically poised (H3K4me1 positive and H3K27ac negative) during pluripotency but become activated upon differentiation as indicated by H3K27ac acquisition .

When analyzing ChIP-seq data, consider that ESRRB may have cell cycle-dependent binding patterns, with enriched binding during G2/M phases of the cell cycle .

What is the role of ESRRB in cancer biology, particularly in breast cancer research?

ESRRB has emerging significance in cancer biology, with notable findings in breast cancer research:

• Expression patterns: ESRRB mRNA expression is significantly lower in Triple Negative Breast Cancer (TNBC)/Basal-Like Breast Cancer (BLBC) compared to other breast cancer subtypes. This decreased expression is not due to copy number loss, as >75% of patients show copy number gain rather than loss at the ESRRB locus (14q24.3) .

• Isoform complexity: ESRRB message is alternatively spliced into three isoforms with different transcription factor activities in basal-like versus other TNBC cell lines. The ERRβ2 and ERRβsf isoforms are broadly expressed in breast tumors at the protein level .

• Functional implications: A small molecule agonist ligand for ESRRB has been reported to have growth inhibitory and anti-mitotic activity in TNBC cell lines, suggesting therapeutic potential .

• Gene correlations: ESRRB mRNA expression correlates with genes associated with neuroactive ligand-receptor interaction, metabolic pathways, and deafness. These genes contain G/C-rich transcription factor binding motifs .

When studying ESRRB in cancer contexts, researchers should employ isoform-specific approaches, as different isoforms display distinct patterns of subcellular localization and transcription factor activity, particularly in TNBC/BLBC .

How does ESRRB function in trophoblast stem cell self-renewal and differentiation?

ESRRB plays critical roles in trophoblast stem cell (TSC) biology:

• Maintenance of self-renewal: ESRRB can block rapid differentiation of TSCs in the absence of Fgf4 and enable accelerated proliferation. It upregulates self-renewal markers (Cdx2, Eomes, Elf5) while inhibiting expression of differentiation markers (Gcm1, Mash2, Tpbpa) .

• Direct transcriptional regulation: ESRRB directly binds and activates a core set of TSC-specific genes and signals including Cdx2, Eomes, Sox2, BMP4, and Fgfr4 .

• Reprogramming capacity: ESRRB can facilitate the conversion of induced TSCs (iTSCs) from Mouse Embryonic Fibroblasts (MEFs). It can substitute for Eomes and initiate iTSC reprogramming along with Gata3, Tfap2c, and c-Myc .

• Cell-autonomous mechanism: Ectopic expression of ESRRB can dramatically increase TSC-like colony numbers by 8-10 times during reprogramming, although it does not obviously shorten the minimal period needed to generate these induced cells .

• Surface marker modulation: During reprogramming, ESRRB expression increases the percentage of CD40-positive cells (a TSC surface marker) more rapidly while decreasing MEF surface marker Thy1 .

Understanding these mechanisms provides insights into placental development and potential therapeutic strategies for placental disorders.

What are the optimal protocols for using ESRRB Antibody, HRP conjugated in ELISA applications?

For optimal ELISA performance using ESRRB Antibody, HRP conjugated:

• Plate preparation:

  • Coat high-binding 96-well plates with target antigen (recombinant ESRRB or cell/tissue lysates) at 1-10 μg/ml in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

  • Wash 3 times with PBST (PBS + 0.05% Tween-20)

  • Block with 1-5% BSA in PBST for 1-2 hours at room temperature

• Antibody application:

  • Prepare serial dilutions of ESRRB Antibody, HRP conjugated (starting from 1:500 to 1:10,000)

  • Add 100 μl of diluted antibody to each well

  • Incubate for 1-2 hours at room temperature with gentle shaking

  • Wash 5 times with PBST to remove unbound antibody

• Detection:

  • Add 100 μl of TMB substrate solution

  • Incubate for 15-30 minutes in the dark (monitor color development)

  • Stop reaction with 50 μl of 2N H₂SO₄

  • Read absorbance at 450 nm with reference at 630 nm

• Controls:

  • Include wells without antigen (coating control)

  • Include wells without primary antibody (secondary antibody background)

  • Include positive control (known ESRRB-expressing sample)

  • Consider using recombinant ESRRB protein for standard curve generation

How can researchers validate the specificity of ESRRB Antibody, HRP conjugated?

Validation of ESRRB Antibody, HRP conjugated specificity is critical and should include:

• Western blot verification:

  • Compare lysates from cells with known ESRRB expression versus ESRRB knockout cells

  • Look for a single band at the expected molecular weight (~55-60 kDa for full-length ESRRB)

  • Perform peptide competition assay using the immunogen peptide (432-502AA region)

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP using the unconjugated version of the ESRRB antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Confirm ESRRB as the predominant protein identified

• Tissue/cell type specificity:

  • Test antibody on tissues with known differential expression of ESRRB

  • Compare results with ESRRB mRNA expression data

  • Consider using cells with CRISPR/Cas9 knockout of ESRRB as negative controls

• Isoform discrimination:

  • Test against different ESRRB isoforms (ERRβ2 and ERRβsf)

  • Compare with isoform-specific antibodies (e.g., ERRβ-clone 07 for ERRβ2 and ERRβ-clone 05 for ERRβsf)

  • Validate subcellular localization patterns match reported nuclear and cytoplasmic distributions

• Cross-reactivity assessment:

  • Test against related nuclear receptors (especially ESRRA and ESRRG)

  • Evaluate reactivity with recombinant proteins from other species to confirm human specificity

What troubleshooting strategies should be employed when experiencing weak or no signal with ESRRB Antibody, HRP conjugated?

When facing weak or absent signals with ESRRB Antibody, HRP conjugated, consider these systematic troubleshooting approaches:

• Antibody activity:

  • Check storage conditions – improper storage or excessive freeze-thaw cycles can diminish activity

  • Verify HRP enzyme activity using a simple substrate test

  • Consider using a fresh aliquot or new lot of antibody

  • Check antibody expiration date

• Antigen retrieval and processing:

  • For protein samples, ensure proper extraction using buffers containing appropriate detergents

  • For ELISA applications, optimize coating conditions including buffer pH and concentration

  • Test different sample preparation methods that preserve ESRRB epitopes

• Detection system:

  • Use enhanced chemiluminescent (ECL) substrates with higher sensitivity

  • Optimize substrate incubation time to prevent signal saturation or depletion

  • Consider using signal amplification systems (e.g., tyramide signal amplification)

• Experimental conditions:

  • Optimize antibody concentration by testing serial dilutions

  • Adjust incubation times and temperatures

  • Use ESRRB-overexpressing cells as positive controls

  • Consider the cell cycle phase of your samples, as ESRRB expression is higher during G2/M phase

• Isoform considerations:

  • Be aware that ESRRB has multiple isoforms with different expression patterns

  • Verify which isoform(s) are expressed in your experimental system

  • The antibody recognizes amino acids 432-502, so confirm this region is present in your target

How is ESRRB expression and function regulated during cell cycle progression?

Recent evidence points to cell cycle-dependent regulation of ESRRB:

• G2/M phase upregulation: ESRRB has been identified as a key pluripotency factor that is upregulated during the G2/M phase of the cell cycle .

• Differentiation control: As a G2/M-specific factor, ESRRB acts as a central driver of extra-embryonic endoderm (XEN) differentiation. Overexpression of ESRRB-YFP in G1 cells can induce XEN differentiation, demonstrating its cell cycle-dependent role in cell fate determination .

• Chromatin occupancy: ChIP-seq analysis of ESRRB in S/G2-enriched embryonic stem cells has revealed binding to enhancers of XEN marker genes (Gata6, Gata4, Foxa2, Dab2, and Foxq1). These enhancers are poised (H3K4me1 positive and H3K27ac negative) during pluripotency but become activated upon differentiation .

• Functional validation: CRISPR/Cas9-mediated knockout of ESRRB prevents XEN differentiation, confirming its necessity for this differentiation pathway .

These findings suggest that researchers should consider cell cycle phase when studying ESRRB expression patterns and functions. Synchronization of cells or cell cycle sorting might be necessary for consistent results when analyzing ESRRB-dependent processes.

What are the differences in ESRRB isoform expression and function across different tissue and cancer types?

ESRRB exhibits complex isoform-specific expression and function patterns:

• Isoform structure: ESRRB message is alternatively spliced into three distinct isoforms, each with different transcription factor activities .

• Cancer-specific patterns:

  • In breast cancer, ERRβ2 and ERRβsf isoforms show distinct patterns of subcellular localization and transcription factor activity, particularly in TNBC/BLBC compared to other subtypes

  • ESRRB mRNA expression is significantly lower in TNBC/BLBC versus other breast cancer subtypes

• Subcellular localization: Immunohistochemistry studies have revealed different patterns of nuclear versus cytoplasmic localization for different isoforms, which may relate to their function .

• Transcriptional activity: The different ESRRB isoforms display varying transcriptional activities:

  Isoform	Transcriptional Activity	Cell Type Specificity
  ERRβ2	Distinct activity pattern	Differential activity in basal-like vs. other TNBC cells
  ERRβsf	Distinct activity pattern	Broadly expressed in breast tumors

• Clinical correlations: Statistical analysis of tissue microarray data has examined ERRβ isoform expression in relation to receptor subtypes, lymph node status, and demographic variables .

These findings highlight the importance of isoform-specific approaches when studying ESRRB in different contexts, as the function of this nuclear receptor appears to be highly context-dependent.

How can ESRRB Antibody, HRP conjugated be optimized for use in multiplex immunoassays?

Incorporating ESRRB Antibody, HRP conjugated into multiplex immunoassays requires careful consideration of several technical aspects:

• Cross-reactivity elimination:

  • Perform extensive antibody validation to ensure no cross-reactivity with other targets in your multiplex panel

  • Use appropriate blocking reagents (5% BSA or commercial blockers with heterophilic antibody blockers)

  • Consider sequential rather than simultaneous detection if cross-reactivity is observed

• Signal discrimination:

  • When using multiple HRP-conjugated antibodies, employ tyramide signal amplification with different fluorophores

  • Consider using different substrates with distinct spectral properties for colorimetric detection

  • Establish optimal dilution for each antibody in the multiplex setting, which may differ from single-target assays

• Optimization strategies:

  • Adjust antibody concentration to achieve comparable signal intensity across all targets

  • Calibrate with recombinant ESRRB protein to establish standard curves

  • Implement stringent washing protocols to minimize background signal

  • Use spike-in controls to assess recovery of each target in the multiplex format

• Compatible detection systems:

  • For microplate-based multiplex assays, pair HRP with appropriate chemiluminescent substrates

  • For tissue-based multiplex assays, consider tyramide signal amplification methods

  • For bead-based multiplex assays, ensure the HRP activity is not affected by coupling chemistry

These considerations will help ensure reliable and specific detection of ESRRB in complex multiplex immunoassay systems while maximizing signal-to-noise ratio.

What approaches can be used to quantify ESRRB isoform-specific expression patterns in tissue samples?

Quantifying ESRRB isoform-specific expression in tissues requires specialized methodological approaches:

• Isoform-specific immunohistochemistry (IHC):

  • Use isoform-specific antibodies (e.g., ERRβ-clone 07 for ERRβ2 and ERRβ-clone 05 for ERRβsf)

  • Implement semi-automatic quantification using digital pathology software

  • Score both nuclear and cytoplasmic staining separately, as different isoforms have distinct subcellular localization patterns

  • Calculate the ratio between different isoforms as this may have biological significance

• Statistical approaches for IHC data:

  • Before statistical analysis, manually examine individual cores and omit any with >50% missing tissue

  • Average scores from multiple cores from each patient to increase reliability

  • For continuous variables, use mean (standard deviation) and median (interquartile range)

  • Apply logit transformation of staining scores to achieve approximate normality

  • Use two-way ANOVA to assess expression differences among different subtypes while considering variables like lymph node status, race, and age

• Correlation analysis:

  • Calculate Spearman's correlation coefficient to measure the association between nuclear and cytoplasmic staining in each subtype

  • Perform pairwise comparisons using Dwass, Steel, Critchlow-Fligner multiple comparison procedure when significant differences are observed

• mRNA-based methods:

  • Design isoform-specific primers for RT-qPCR

  • Use RNAseq with specific analysis pipelines for alternative splicing detection

  • Consider digital droplet PCR for absolute quantification of low-abundance isoforms