ESRRG Antibody

What is ESRRG and why is it important in research applications?

ESRRG (estrogen-related receptor gamma), also known as NR3B3 (nuclear receptor subfamily 3, group B, member 3), is a member of the nuclear hormone receptor family of steroid hormone receptors. As an orphan nuclear receptor, it binds specifically to estrogen response elements and activates reporter genes . ESRRG has significant research importance due to its roles in various biological processes, including renal development and metabolic regulation. Recent studies have demonstrated its involvement in suppressing esophageal squamous cell carcinoma (ESCC) through metabolic reprogramming . The protein's molecular weight is approximately 51 kDa, and it functions as a transcriptional regulator with implications in development, metabolism, and disease processes .

What applications are ESRRG antibodies validated for in research settings?

ESRRG antibodies have been validated for multiple research applications, with varying degrees of effectiveness depending on the specific antibody and target tissue. The primary validated applications include:

• Western Blot (WB): Typically used at dilutions between 1:1000-1:4000, with positive detection in human spleen and kidney tissues

• Immunohistochemistry (IHC): Effective at dilutions of 1:2000-1:8000, with positive detection in rat stomach tissue, mouse and rat brain tissue, and mouse and rat heart tissue

• Immunofluorescence (IF): Validated for cellular localization studies

• ELISA: Used for quantitative protein detection

• Chromatin Immunoprecipitation (ChIP): Some ESRRG antibodies are specifically validated for studying protein-DNA interactions

For optimal results, researchers should titrate these antibodies in their specific testing systems, as effectiveness can be sample-dependent .

What is the optimal protocol for immunohistochemical detection of ESRRG?

For effective immunohistochemical detection of ESRRG, follow this methodological approach:

• Sample preparation: Use 4-6 μm sections from paraformaldehyde-fixed, paraffin-embedded tissue samples.

• Antigen retrieval:

  • Primary method: Heat sections in TE buffer (pH 9.0) using a pressure cooker for 4 minutes

  • Alternative method: Boil sections in 10 mM citrate buffer (pH 6.0) twice for 30 seconds and allow to cool in buffer for 20 minutes

• Blocking: Apply 10% heat-inactivated serum (sheep or appropriate species) in PBS to reduce non-specific binding.

• Primary antibody incubation: Dilute ESRRG antibody to 1:500-1:2000 (depending on specific antibody) and incubate at 4°C overnight in a humidified chamber .

• Secondary antibody application: Apply biotinylated anti-rabbit IgG (1:1000) for 1 hour at room temperature.

• Detection: Use either:

  • Vector Lab ABC kit with NBT/BCIP and counterstain with eosin

  • 3,3′-diaminobenzidine tetrahydrochloride, assessing expression based on staining intensity and extent

For more complex co-localization studies, consider dual immunofluorescence with appropriate tissue-specific markers to contextualize ESRRG expression patterns.

How can I address non-specific binding when using ESRRG antibodies?

Non-specific binding is a common challenge when working with ESRRG antibodies. To address this issue, implement these methodological approaches:

• Optimize blocking conditions: Increase blocking serum concentration to 10-15% and extend the blocking time to 1-2 hours at room temperature. Consider using the serum from the same species as your secondary antibody .

• Antibody dilution optimization: Titrate your antibody across a broader range than recommended (e.g., 1:500 to 1:8000) to identify the optimal signal-to-noise ratio for your specific tissue or cell type .

• Buffer modifications: Add 0.1-0.3% Triton X-100 to your antibody dilution buffer to reduce background in IHC/IF applications. For western blots, include 0.1% Tween-20 in washing buffers.

• Include validated controls:

  • Positive controls: Human kidney or spleen tissue for WB; rat brain or heart tissue for IHC

  • Negative controls: Omit primary antibody on duplicate sections

  • Specificity controls: Consider using ESRRG knockout or knockdown samples if available

• Cross-adsorption: If cross-reactivity is suspected, pre-adsorb the antibody with the immunizing peptide or irrelevant proteins to remove non-specific antibodies.

By systematically implementing these approaches, researchers can significantly improve the specificity of ESRRG antibody staining across different experimental platforms.

Why might ESRRG antibody detection fail in Western blot applications?

Failed detection of ESRRG in Western blot experiments can stem from multiple methodological issues. Consider these troubleshooting approaches:

• Sample preparation concerns:

  • Ensure complete protein denaturation using appropriate SDS concentration and heating (95°C for 5 minutes)

  • Verify protein extraction efficiency from nuclear fractions, as ESRRG is a nuclear receptor

  • Add protease inhibitors freshly to prevent degradation

• Transfer optimization:

  • For the 51 kDa ESRRG protein, use PVDF membranes with 0.45 μm pore size

  • Consider semi-dry transfer at 15V for 30-45 minutes or wet transfer at 30V overnight at 4°C

  • Verify transfer efficiency using reversible protein stains (Ponceau S)

• Antibody considerations:

  • Start with the recommended 1:1000-1:4000 dilution range, adjusting based on signal strength

  • Extend primary antibody incubation to overnight at 4°C

  • Ensure antibody recognizes denatured epitopes (some antibodies only work in native conditions)

• Detection system sensitivity:

  • If expression is low, switch to more sensitive chemiluminescent substrates

  • Consider amplification steps like biotin-streptavidin systems

  • Increase exposure time incrementally to detect weak signals

• Control experiments:

  • Run positive control tissues known to express ESRRG (human kidney or spleen)

  • Include recombinant ESRRG protein as a standard if available

Remember that the observed molecular weight of ESRRG is approximately 51 kDa , but post-translational modifications may alter the apparent molecular weight in different tissue contexts.

How can I design experiments to investigate ESRRG's role in metabolic reprogramming?

To investigate ESRRG's role in metabolic reprogramming, particularly in cancer contexts, design your experimental approach using these methodological strategies:

• Genetic manipulation models:

  • Generate stable ESRRG-overexpressing cell lines using lentiviral vectors at a multiplicity of infection of 35 plaque-forming units per cell

  • Create ESRRG knockdown models using shRNA or siRNA approaches

  • Select stable transgenic cells with puromycin (1 μg/ml) and validate expression changes via Western blot and qRT-PCR

• Metabolic phenotyping:

  • Measure glycolytic parameters (glucose uptake, lactate production, extracellular acidification rate)

  • Assess mitochondrial function (oxygen consumption rate, ATP production)

  • Analyze expression of key glycolytic enzymes, particularly PKM2, which has been shown to interact with ESRRG

• Molecular interaction studies:

  • Perform chromatin immunoprecipitation (ChIP) to identify ESRRG binding to promoters of metabolic genes

  • Use reporter assays to assess ESRRG's effect on promoter activity of target genes

  • Conduct co-immunoprecipitation experiments to identify protein-protein interactions in the metabolic regulatory network

• Pharmacological approaches:

  • Test the ESRRG-specific agonist DY131 to modulate ESRRG activity and observe effects on metabolic parameters

  • Compare results with genetic manipulation approaches to validate mechanism specificity

• Transcriptomic profiling:

  • Perform RNA-Seq on control vs. ESRRG-manipulated cells

  • Conduct Gene Set Enrichment Analysis (GSEA) with a focus on metabolic pathways

  • Validate key differentially expressed genes with qRT-PCR

This comprehensive experimental approach will provide mechanistic insights into how ESRRG regulates metabolic reprogramming, particularly in disease contexts such as cancer.

What are the best methods for studying ESRRG's interaction with the PKM2 promoter?

To effectively study ESRRG's interaction with the PKM2 promoter, implement these specialized methodological approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA interactions with 1% formaldehyde for 10 minutes

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate using validated ESRRG antibodies

  • Design primers targeting the PKM2 promoter region for qPCR analysis

  • Include positive controls (known ESRRG targets) and negative controls (non-target regions)

• Promoter reporter assays:

  • Clone the PKM2 promoter region into a luciferase reporter vector

  • Create deletion and point mutation constructs to identify specific ESRRG binding elements

  • Co-transfect with ESRRG expression vectors and measure luciferase activity

  • Include appropriate controls (empty vectors, unrelated transcription factors)

• DNA-protein binding assays:

  • Perform Electrophoretic Mobility Shift Assays (EMSA) using nuclear extracts from ESRRG-expressing cells

  • Include competitive and non-competitive unlabeled probes to confirm binding specificity

  • Use supershift assays with ESRRG antibodies to verify complex formation

• In vivo footprinting:

  • Apply in vivo DNase I footprinting to identify protected regions within the PKM2 promoter

  • Compare footprints between ESRRG-expressing and non-expressing cells

• Integrative genomic approaches:

  • Combine ChIP-seq data for ESRRG with open chromatin assays (ATAC-seq)

  • Correlate with RNA-seq data to establish functional consequences of binding

  • Validate using targeted approaches like CRISPRi to disrupt specific binding sites

This multi-faceted approach will provide robust evidence of direct ESRRG regulation of the PKM2 promoter, as suggested by existing research showing that ESRRG directly interacts with the PKM2 promoter to inhibit its activity in esophageal squamous cell carcinoma .

What are the optimal conditions for detecting ESRRG in renal development studies?

For detecting ESRRG in renal development studies, optimize your experimental approach using these methodological considerations:

• Developmental stage selection:

  • Focus on early stages of ureteric bud development, as ESRRG functions in early branch generation

  • Include samples from multiple developmental timepoints (E11.5-E18.5 in mice) to track expression changes

• Tissue preparation optimizations:

  • Use paraformaldehyde fixation (4% for 24 hours) for embryonic tissues

  • Consider vibratome sectioning (100-150 μm) for 3D analysis of branching structures

  • For paraffin sections, use 4-6 μm thickness with careful orientation to capture branching patterns

• Antigen retrieval specifics:

  • For embryonic kidney tissues, use citrate buffer (pH 6.0) with two 30-second microwave heating cycles

  • Allow sections to cool in buffer for 20 minutes to optimize epitope exposure

• Antibody selection and dilution:

  • Use antibodies validated specifically for developmental studies

  • Begin with 1:500 dilution and optimize based on signal-to-noise ratio

  • Consider using rabbit polyclonal antibodies which may provide better sensitivity for low-abundance developmental expression

• Co-localization studies:

  • Pair ESRRG staining with ureteric bud markers (e.g., Cdh1) and metanephric mesenchyme markers (e.g., Six2)

  • Use fluorescent secondary antibodies for multi-color analysis of expression domains

  • Consider confocal microscopy for detailed spatial resolution

• Complementary techniques:

  • Validate IHC findings with in situ hybridization for ESRRG mRNA

  • Consider laser capture microdissection followed by qRT-PCR for quantitative analysis of expression in specific structures

These optimized conditions will facilitate accurate detection of ESRRG during renal development, enabling proper characterization of its role in ureteric bud branching and renal papilla formation .

How can I effectively detect ESRRG in cardiac tissue samples?

For optimal detection of ESRRG in cardiac tissue samples, implement these methodological approaches tailored to the unique challenges of cardiac tissue:

• Tissue preservation considerations:

  • For fresh cardiac tissue, fix in 4% paraformaldehyde for 24-48 hours

  • For archived samples, ensure proper paraffin embedding with controlled orientation

  • Consider using mouse or rat heart tissues which show positive IHC detection with ESRRG antibodies

• Section preparation specifics:

  • For paraffin sections, cut at 4-6 μm thickness

  • For frozen sections, cut at 8-10 μm and fix briefly in acetone or 4% PFA

  • Mount on positively charged slides to prevent tissue loss during processing

• Optimized antigen retrieval:

  • Primary method: Use TE buffer (pH 9.0) for heat-induced epitope retrieval

  • Alternative: Citrate buffer (pH 6.0) with controlled heating cycles

  • Allow 20-30 minutes of cooling in retrieval buffer

• Blocking and permeabilization:

  • Block with 10% serum (matching secondary antibody species)

  • Add 0.1-0.3% Triton X-100 for improved antibody penetration

  • Consider adding 1% BSA to reduce background in cardiac tissue

• Antibody application specifics:

  • Primary antibody: Use at 1:2000-1:8000 dilution for IHC applications

  • Incubation: Overnight at 4°C in a humidified chamber

  • Secondary antibody: 1:1000 dilution for 1 hour at room temperature

• Detection and counterstaining:

  • DAB development: Monitor closely as cardiac tissue can develop high background

  • Counterstain: Use hematoxylin briefly (30 seconds) to avoid masking specific signals

  • Mounting: Use permanent mounting media for long-term storage

• Controls and validation:

  • Include positive controls (known ESRRG-expressing cardiac regions)

  • Use ESRRG knockout tissues as negative controls if available

  • Compare with published expression patterns in mouse and rat heart tissues

These methodological refinements will enhance the specificity and sensitivity of ESRRG detection in cardiac tissues, facilitating accurate characterization of its expression patterns and functional significance in cardiac biology.

How can I design experiments to investigate ESRRG's therapeutic potential through agonists like DY131?

To investigate ESRRG's therapeutic potential through agonists like DY131, implement this comprehensive experimental design:

• In vitro efficacy assessment:

  • Establish dose-response curves (typically 0.1-100 μM) in relevant cell lines

  • Measure effects on cell proliferation, metabolism, and specific pathway activation

  • Compare DY131 effects in ESRRG-overexpressing versus knockdown cells to confirm specificity

  • Assess combination effects with standard therapeutic agents

• Mechanistic investigation:

  • Perform RNA-Seq on control versus DY131-treated cells to identify transcriptional changes

  • Conduct metabolomic profiling to characterize changes in glycolysis and other metabolic pathways

  • Use GSEA to identify enriched pathways after DY131 treatment

  • Validate key targets using qRT-PCR and Western blotting

• Pharmacodynamic marker development:

  • Identify and validate biomarkers that reflect DY131 activity on ESRRG

  • Develop assays to measure PKM2 expression/activity as a direct marker of ESRRG activation

  • Establish time course of biomarker changes after DY131 administration

• Preclinical models:

  • Test DY131 in appropriate disease models (e.g., patient-derived xenografts for ESCC)

  • Determine optimal dosing regimens (dose, schedule, route of administration)

  • Measure tumor growth inhibition, metabolic changes, and immune microenvironment alterations

  • Assess toxicity and pharmacokinetic properties

• Immune checkpoint interaction studies:

  • Investigate DY131's effect on immune checkpoint pathways

  • Test combination therapies with established immune checkpoint inhibitors

  • Analyze changes in tumor microenvironment after DY131 treatment

  • Measure lactate levels as a mediator of immune suppression

This comprehensive experimental approach will establish the therapeutic potential of DY131 and other ESRRG agonists, with particular emphasis on metabolic reprogramming and immune modulation in cancer contexts.

What approaches can I use to study the role of ESRRG in cross-talk between metabolic regulation and immune response?

To investigate ESRRG's role in the cross-talk between metabolic regulation and immune response, implement these advanced methodological approaches:

• Co-culture experimental systems:

  • Establish co-cultures of ESRRG-manipulated cancer cells with immune cells (T cells, macrophages)

  • Measure immune cell activation, proliferation, and effector functions

  • Analyze metabolite exchange using isotope tracing or metabolomic approaches

  • Test the impact of DY131 treatment on immune cell function in co-culture systems

• Lactate pathway analysis:

  • Quantify lactate production in ESRRG-overexpressing versus control cells

  • Measure expression of lactate transporters (MCTs) and lactate receptor GPR81

  • Assess the impact of exogenous lactate on immune cell function

  • Test whether ESRRG-mediated suppression of PKM2 reduces lactate production and enhances immune cell activity

• Immune checkpoint regulation:

  • Analyze the expression of immune checkpoint molecules (PD-L1, CTLA-4) in response to ESRRG modulation

  • Use flow cytometry to measure checkpoint molecule expression on tumor and immune cells

  • Test whether DY131 enhances the activity of immune checkpoint inhibitors

  • Perform RNA-Seq to identify transcriptional networks linking ESRRG to immune regulation

• In vivo immune microenvironment studies:

  • Establish syngeneic mouse models with ESRRG-modulated tumors

  • Characterize tumor-infiltrating immune populations by flow cytometry and immunohistochemistry

  • Test combination therapies with DY131 and immune checkpoint inhibitors

  • Perform single-cell RNA-Seq to define cell-type-specific responses

• Mechanistic dissection using CRISPR screens:

  • Conduct CRISPR screens targeting metabolic and immune pathway genes in ESRRG-modulated cells

  • Identify synthetic lethal interactions that could be therapeutically targeted

  • Validate key nodes using individual gene knockouts and rescue experiments

This multifaceted approach will elucidate how ESRRG-mediated metabolic reprogramming influences anti-tumor immunity, potentially revealing new therapeutic strategies that leverage this cross-talk for enhanced efficacy of immunotherapies .

How do different ESRRG antibodies compare in terms of epitope recognition and application suitability?

Different ESRRG antibodies demonstrate variable performance characteristics based on their epitope recognition and production methods. This comparative analysis provides guidance for application-specific selection:

When selecting an ESRRG antibody:

• Consider epitope accessibility: N-terminal antibodies often perform better in native protein detection, while middle or C-terminal antibodies may be superior for denatured applications.

• Application-specific considerations:

  • For ChIP applications: N-terminal antibodies show superior performance

  • For IHC: Middle region antibodies provide more consistent results across species

  • For quantitative assays: C-terminal antibodies offer better linearity in ELISA applications

• Validation method alignment: Select antibodies validated using methods similar to your intended application, as performance can vary significantly across techniques.

This comparative analysis highlights the importance of selecting application-appropriate ESRRG antibodies based on experimental goals and target species.

How should I interpret conflicting ESRRG expression data between different experimental techniques?

When faced with conflicting ESRRG expression data across different experimental techniques, implement this systematic analytical approach:

• Technical validation assessment:

  • Verify antibody specificity through knockout/knockdown controls for protein-based methods

  • For RNA-based methods, check primer specificity and efficiency

  • Consider whether each method measures different aspects (mRNA vs. protein, total vs. active protein)

• Methodological sensitivity comparison:

  • Western blot: Typically detects ESRRG at expected 51 kDa molecular weight

  • IHC/IF: Provides spatial information but may have threshold detection limitations

  • qRT-PCR: Highly sensitive for transcript detection but doesn't reflect protein levels

  • RNA-Seq: Provides comprehensive transcriptomic context but requires proper normalization

• Sample preparation differences:

  • Consider whether nuclear extraction was performed (critical for nuclear receptors like ESRRG)

  • Evaluate fixation effects on epitope availability in IHC/IF

  • Assess RNA quality metrics (RIN values) for transcript analyses

• Biological context analysis:

  • Developmental stage differences may explain discrepancies (ESRRG functions in early development)

  • Metabolic state of cells/tissues can alter ESRRG expression and localization

  • Disease context may influence expression (e.g., reduced in ESCC compared to normal tissue)

• Resolution framework:

  • Prioritize data from methods with appropriate controls

  • Consider orthogonal validation using alternative antibodies or detection methods

  • When possible, use genetic approaches (overexpression, knockdown) to validate functional relevance

  • Employ single-cell approaches to resolve potential cellular heterogeneity issues

By systematically analyzing these factors, researchers can resolve apparently conflicting data and develop a more nuanced understanding of ESRRG expression patterns across different experimental contexts.

What emerging methodologies might enhance our ability to study ESRRG's tissue-specific functions?

Several cutting-edge methodologies are poised to revolutionize our understanding of ESRRG's tissue-specific functions:

• Spatial transcriptomics and proteomics:

  • Technologies like Visium and GeoMx DSP can map ESRRG expression with spatial context

  • These approaches reveal microenvironmental influences on ESRRG function

  • Integration with single-cell data provides unprecedented resolution of heterogeneous responses

  • Particularly valuable for developmental contexts where ESRRG shows spatially restricted expression patterns

• CRISPR-based functional genomics:

  • CRISPR activation/interference systems allow tissue-specific modulation of ESRRG

  • Base editing technologies enable precise modification of ESRRG binding sites

  • CRISPR screens can identify tissue-specific cofactors and downstream effectors

  • In vivo CRISPR delivery methods permit temporal control of ESRRG function

• Organoid and tissue-on-chip models:

  • Organ-specific 3D cultures better recapitulate in vivo ESRRG function

  • Patient-derived organoids allow study of ESRRG in human disease contexts

  • Microfluidic organ-on-chip systems enable analysis of metabolic functions in controlled environments

  • Co-culture systems can investigate ESRRG's role in cellular cross-talk

• Integrative multi-omics approaches:

  • Combined analysis of transcriptomics, proteomics, and metabolomics data

  • Chromatin conformation capture techniques (Hi-C, HiChIP) to map 3D genomic interactions

  • Computational modeling of ESRRG-regulated metabolic networks

  • Systems biology approaches to contextualize ESRRG within tissue-specific regulatory networks

• Advanced imaging technologies:

  • CODEX multiplexed protein imaging for simultaneous detection of multiple markers

  • Intravital microscopy to observe ESRRG function in living tissues

  • Super-resolution techniques to visualize subcellular localization and dynamics

  • Label-free imaging of metabolites to directly visualize ESRRG's impact on metabolism

These emerging methodologies will provide unprecedented insights into ESRRG's tissue-specific functions, particularly in developmental contexts and metabolic regulation, advancing our understanding of this important nuclear receptor.

What are the key unanswered questions regarding ESRRG antibodies and their research applications?

Several critical questions remain unanswered regarding ESRRG antibodies and their research applications, presenting opportunities for methodological advancement and biological discovery:

• Isoform-specific detection challenges:

  • How can we develop and validate antibodies that specifically recognize distinct ESRRG isoforms?

  • What are the functional differences between ESRRG isoforms across tissues and developmental stages?

  • How do post-translational modifications affect epitope recognition and antibody performance?

• Dynamic regulation visualization:

  • How can we develop tools to visualize ESRRG activity in live cells and tissues?

  • What approaches would enable temporal tracking of ESRRG nuclear translocation and DNA binding?

  • How might biosensor development advance our understanding of ESRRG activation dynamics?

• Therapeutic targeting considerations:

  • How do ESRRG agonists like DY131 affect antibody binding characteristics?

  • Can we develop antibody-based tools to assess agonist engagement with ESRRG in vivo?

  • What biomarkers might predict response to ESRRG-targeted therapeutics?

• Methodological standardization needs:

  • What reference standards should be adopted for ESRRG antibody validation?

  • How can we develop consensus protocols for tissue-specific applications?

  • What reporting standards would improve reproducibility in ESRRG antibody-based research?

• Emerging application frontiers:

  • How might single-cell proteomics techniques be adapted for ESRRG detection?

  • What multiplex imaging approaches could contextualize ESRRG within regulatory networks?

  • How can proximity labeling approaches identify novel ESRRG interaction partners?

• Cross-species considerations:

  • How conserved are ESRRG epitopes across species, and how does this affect antibody selection?

  • What reference tissues should be used for validation in different model organisms?

  • How can we better translate findings between model systems and human diseases?

Addressing these outstanding questions will advance not only our technical capabilities but also our fundamental understanding of ESRRG biology, potentially revealing new therapeutic opportunities in development, metabolism, and disease.