RORA Antibody

What is RORA and why are antibodies against it important for research?

RORA (RAR-related orphan receptor A) is a nuclear receptor protein of approximately 59 kilodaltons that functions as a transcription factor. It is also known by several alternative names including ROR3, RZRA, sg, IDDECA, NR1F1, and nuclear receptor ROR-alpha . RORA antibodies are critical research tools for studying this protein's role in circadian rhythm regulation, metabolism, immune function, and neurological development. The significance of these antibodies lies in their ability to selectively bind to RORA proteins across various experimental applications, allowing researchers to investigate expression patterns, protein-protein interactions, and functional roles in different physiological and pathological contexts .

What are the structural domains of RORA that antibodies typically target?

RORA antibodies are designed to target specific domains of the protein, with each targeting strategy offering distinct experimental advantages. The most commonly targeted regions include:

• N-terminal domain (N-Term): Critical for specificity determination

• Middle region: Important for functional studies of the central portion of the protein

• Hinge region: Allows flexibility between DNA-binding and ligand-binding domains

• Ligand-binding domain: Essential for studying RORA's interaction with various ligands

The selection of antibodies targeting specific domains should align with research questions - for instance, studies investigating ligand interactions benefit from antibodies targeting the ligand-binding domain, while transcriptional regulation studies might utilize antibodies targeting DNA-binding domains.

What species reactivity should researchers consider when selecting RORA antibodies?

When selecting RORA antibodies, researchers must carefully evaluate species reactivity based on their experimental model. Available RORA antibodies demonstrate varying cross-reactivity profiles:

Researchers must verify the sequence homology between their study species and the immunogen used to develop the antibody, particularly when working with less common model organisms .

What are the validated applications for RORA antibodies in research settings?

RORA antibodies have been validated for multiple experimental applications, each requiring specific technical considerations:

Researchers should validate each application independently, as performance in one application does not guarantee success in another. Optimization experiments are essential for each new cell line, tissue type, or experimental condition .

How should researchers optimize western blotting protocols specifically for RORA detection?

For optimal western blot detection of RORA, researchers should consider these methodological recommendations:

• Sample preparation: Use RIPA or NP-40 lysis buffers with protease inhibitors, ensuring nuclear fraction retention since RORA is a nuclear receptor.

• Gel selection: 8-10% acrylamide gels are optimal for resolving the ~59 kDa RORA protein.

• Transfer conditions: Semi-dry or wet transfer at 100V for 60-90 minutes using PVDF membranes (preferred over nitrocellulose for nuclear proteins).

• Blocking: 5% BSA in TBST is generally more effective than milk-based blockers for nuclear protein detection.

• Primary antibody incubation: Overnight at 4°C using specific anti-RORA antibodies at optimized dilutions (typically 1:500-1:1000).

• Washing: Extensive washing (4-5 times, 5 minutes each) with TBST to reduce background.

• Controls: Include positive controls (tissue/cells known to express RORA) and negative controls (RORA knockout samples when available).

Validation through multiple antibodies targeting different epitopes is recommended to confirm specificity of bands at the expected molecular weight .

How can RORA antibodies be employed in chromatin immunoprecipitation (ChIP) studies?

For ChIP applications with RORA antibodies, researchers should implement these specialized protocols:

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes works for most applications, but RORA may require optimization between 0.5-2% formaldehyde and 5-15 minutes of crosslinking time.

• Sonication parameters: Nuclear receptors like RORA typically require more stringent chromatin shearing; aim for 200-500bp fragments with 10-15 cycles of sonication (30 seconds on/30 seconds off).

• Antibody selection: Choose ChIP-validated RORA antibodies specifically targeting DNA-binding domains or N-terminal regions for highest efficiency.

• Immunoprecipitation conditions: Pre-clear chromatin with protein A/G beads, then incubate with 3-5μg of RORA antibody overnight at 4°C.

• Washing stringency: Implement progressively stringent washes to reduce background while maintaining specific RORA-DNA complexes.

• Control experiments: Include IgG controls, input controls, and ideally, RORA-depleted negative controls.

• Validation: Confirm enrichment at known RORA binding sites via qPCR before proceeding to genome-wide analyses.

The success of RORA ChIP experiments depends heavily on antibody quality and protocol optimization specific to each cell type and experimental condition .

What considerations are important when using RORA antibodies in co-immunoprecipitation studies?

When performing co-immunoprecipitation (co-IP) to study RORA protein interactions:

• Lysis buffer selection: Use gentle, non-denaturing buffers (e.g., 150mM NaCl, 50mM Tris-HCl pH 7.5, 1% NP-40) supplemented with protease inhibitors and phosphatase inhibitors to preserve protein-protein interactions.

• Nuclear extraction: Implement specialized nuclear extraction protocols since RORA primarily localizes to the nucleus, using low detergent concentrations to preserve protein complexes.

• Pre-clearing strategy: Pre-clear lysates with appropriate control beads to reduce non-specific binding.

• Antibody orientation: Consider both direct immunoprecipitation (using anti-RORA antibodies) and reverse co-IP (using antibodies against suspected interaction partners) for confirmation.

• Crosslinking considerations: Reversible crosslinking agents may help stabilize transient interactions between RORA and its cofactors.

• Validation approaches: Confirm interactions through reciprocal co-IPs and additional techniques such as proximity ligation assays or FRET.

• Controls: Include isotype controls, input samples, and ideally RORA-knockout negative controls.

These methodological considerations help ensure that detected interactions represent true biological associations rather than experimental artifacts .

How can researchers validate the specificity of RORA antibodies in their experimental systems?

Comprehensive validation of RORA antibodies should include multiple complementary approaches:

• Knockout/knockdown controls: The gold standard for antibody validation involves comparing signals between wild-type samples and those where RORA has been genetically deleted or depleted through CRISPR-Cas9, RNAi, or similar technologies.

• Overexpression systems: Complementary to knockout approaches, comparing signals between normal and RORA-overexpressing samples provides additional validation.

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals.

• Multiple antibody concordance: Testing multiple antibodies targeting different RORA epitopes should yield consistent results in terms of localization, molecular weight, and expression patterns.

• Recombinant protein standards: Include purified recombinant RORA protein as a positive control in western blots to confirm correct molecular weight detection.

• Mass spectrometry validation: For definitive confirmation, immunoprecipitate RORA and verify its identity through mass spectrometry.

• Cross-reactivity assessment: Test the antibody in systems where related proteins (e.g., RORB, RORC) are expressed but RORA is absent.

This multi-faceted validation approach ensures reliable results, particularly for advanced applications like quantitative studies or localization experiments .

How do experimental conditions affect RORA antibody performance in immunocytochemistry?

For optimal immunocytochemical detection of RORA, researchers must consider these critical parameters:

• Fixation method impacts:

  • Paraformaldehyde (4%): Preserves morphology but may mask nuclear epitopes

  • Methanol/acetone: Better for nuclear antigen accessibility but compromises membrane integrity

  • Combined approaches: Sequential PFA and methanol fixation often optimal for nuclear receptors like RORA

• Permeabilization optimization:

  • Nuclear proteins require enhanced permeabilization (0.2-0.5% Triton X-100 for 10-15 minutes)

  • Saponin (0.1-0.3%) provides gentler alternative with less epitope disruption

• Antigen retrieval techniques:

  • Heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Enzymatic retrieval (proteinase K treatment)

  • Combinatorial approaches for complex tissues

• Blocking parameters:

  • 5-10% normal serum from secondary antibody host species

  • Addition of 0.1-0.3% Triton X-100 to blocking solution improves nuclear penetration

• Primary antibody incubation:

  • Extended incubation (overnight at 4°C) at optimized dilution

  • Use of antibody diluents containing carrier proteins and stabilizers

• Signal amplification options:

  • Tyramide signal amplification for low abundance detection

  • Indirect detection with specialized secondary antibody systems

Each parameter requires systematic optimization for specific experimental systems to balance signal intensity with specificity .

What are common issues with RORA antibodies in western blotting and how can they be resolved?

Researchers frequently encounter these challenges when using RORA antibodies in western blotting:

RORA detection may be particularly challenging due to its nuclear localization and potential association with chromatin fractions. Optimizing nuclear extraction protocols and including phosphatase inhibitors can help address these specific challenges .

How should researchers interpret discrepancies in RORA detection between different antibodies or techniques?

When encountering discrepancies in RORA detection:

• Epitope accessibility analysis: Different antibodies target distinct epitopes that may be differentially accessible depending on protein conformation, interactions, or modifications. Map the epitopes of each antibody and assess their accessibility in your experimental conditions.

• Isoform specificity evaluation: RORA has multiple isoforms (RORα1, RORα2, RORα3, RORα4) with tissue-specific expression patterns. Determine which isoforms each antibody detects and correlate with expected expression in your system.

• Technical parameters assessment: Each detection method has different sensitivity thresholds and dynamic ranges. Western blotting provides molecular weight information but may miss low-abundance forms, while IHC provides localization but with potential cross-reactivity.

• Post-translational modification consideration: RORA undergoes sumoylation, phosphorylation, and other modifications that can affect antibody recognition. Test whether treatments altering these modifications affect detection patterns.

• Comprehensive validation approach: When discrepancies occur, implement orthogonal methods including:

  • Mass spectrometry for definitive identification

  • Genetic manipulation (overexpression/knockdown)

  • mRNA analysis correlation with protein detection

Researchers should systematically document discrepancies and consider them valuable data points indicating potential biological complexity rather than merely technical failures .

How can RORA antibodies be integrated into single-cell analysis techniques?

Integrating RORA antibodies into emerging single-cell technologies requires specialized approaches:

• Antibody-based single-cell sorting: Flow cytometry using RORA antibodies requires extensive optimization for nuclear proteins, including specialized fixation and permeabilization protocols that maintain cellular integrity while allowing nuclear access.

• Mass cytometry (CyTOF) applications: Metal-conjugated RORA antibodies require validation for epitope preservation following conjugation processes, with careful panel design to avoid signal spillover.

• Single-cell western blotting: Microfluidic platforms for single-cell protein analysis require highly specific antibodies with minimal background, with RORA detection needing specialized nuclear protein extraction protocols.

• Spatial transcriptomics integration: Combining RORA protein detection with mRNA localization through techniques like MERFISH allows correlation between transcription and protein expression at single-cell resolution.

• Automated high-content imaging: Machine learning algorithms for RORA localization and quantification require extensive training with validated antibodies to distinguish specific signals from background.

These advanced approaches benefit from the recent development of recombinant antibody technologies that provide highly consistent lot-to-lot performance for reproducible single-cell studies .

What emerging research areas could benefit from advanced RORA antibody applications?

Several cutting-edge research areas could be advanced through sophisticated RORA antibody applications:

• Circadian biology: RORA's role in circadian rhythm regulation could be investigated through temporal ChIP-seq studies using highly specific antibodies to map dynamic binding patterns throughout the day-night cycle.

• Neuroinflammation research: The interplay between RORA, microglia, and neuroinflammatory processes could be explored using multiplex immunofluorescence with RORA antibodies combined with microglial and inflammatory markers.

• Cancer immunotherapy: RORA's emerging role in T-cell development and function suggests potential for combining RORA antibodies with immunotherapy biomarker panels for patient stratification.

• Metabolic disease mechanisms: RORA's involvement in lipid metabolism could be investigated through proximity ligation assays using RORA antibodies paired with metabolic enzyme antibodies.

• Neurodevelopmental disorder research: Given RORA's implications in autism spectrum disorders, antibodies for developmental studies across brain regions and developmental timepoints could reveal mechanistic insights.

• Environmental toxicology: As an environmental sensor, RORA's response to endocrine-disrupting chemicals could be monitored using reporter assays validated with specific antibodies.

These emerging fields require highly specific antibodies with validated performance in complex biological systems and often benefit from combining antibody-based detection with complementary genomic and functional approaches .

How do next-generation sequencing technologies enhance antibody development for targets like RORA?

Recent innovations in antibody development utilizing NGS technologies offer significant advantages for generating RORA-specific antibodies:

• Genotype-phenotype linkage systems: New screening methods compatible with NGS allow rapid identification of antigen-specific clones through the expression of membrane-bound immunoglobulins, enabling direct functional screening that links antigen binding with DNA sequence information .

• High-throughput functional screening: Unlike conventional hybridoma techniques, NGS-based methods allow for the rapid screening of tens of thousands of antibody candidates against RORA, significantly reducing development time .

• Enhanced diversity capture: NGS-enabled technologies capture a broader antibody repertoire by sequencing numerous B-cell populations, including rare clones that might be overlooked in traditional screening approaches.

• Affinity maturation tracking: Sequential sequencing during immunization allows researchers to track the evolution of antibody affinity, particularly valuable for difficult targets like conformationally sensitive regions of RORA.

• Computational epitope prediction: Integration of sequence data with structural predictions helps identify antibodies targeting functionally relevant epitopes on RORA.

• Single-cell paired heavy and light chain recovery: NGS coupled with single-cell isolation ensures proper pairing of heavy and light chains, preserving natural antibody configurations for optimal binding to RORA.

These technological advances have transformed antibody development from months-long processes to potentially weeks, while simultaneously improving specificity and functionality .

What advantages do recombinant antibodies offer over traditional monoclonal antibodies for RORA research?

Recombinant RORA antibodies provide several significant advantages over traditional hybridoma-derived antibodies:

• Sequence-defined production: Unlike hybridoma-derived antibodies, recombinant antibodies have defined amino acid sequences, eliminating drift in production and ensuring consistent performance across different production lots.

• Engineering flexibility: The defined sequence allows for targeted modifications including:

  • Humanization for therapeutic applications

  • Fragment generation (Fab, scFv) for improved tissue penetration

  • Fusion with reporter proteins for direct visualization

  • Site-specific conjugation for precise labeling

• Reproducibility advantages: Expression in defined systems eliminates hybridoma instability issues and batch-to-batch variation, which is particularly important for quantitative RORA studies.

• Reduced background: Recombinant antibodies can be engineered to minimize cross-reactivity with related nuclear receptors (RORB, RORC), enhancing specificity for RORA.

• Epitope precision: Recombinant technology allows targeting of specific functional domains of RORA with greater precision than conventional approaches.

• Ethical considerations: Development without animal immunization aligns with efforts to reduce animal use in research.

For RORA research specifically, recombinant antibodies with defined binding to specific protein domains facilitate more precise interrogation of RORA's varied functions in different cellular contexts .