rargb Antibody

What is RARGB and what biological systems is it involved in?

RARGB (Retinoic Acid Receptor Gamma B) is a protein-coding gene that belongs to the nuclear hormone receptor superfamily of transcriptional regulators. According to genomic databases, RARGB is predicted to enable RNA polymerase II cis-regulatory region sequence-specific DNA binding activity and nuclear receptor activity . It acts upstream of or within several critical developmental processes, including:

• Determination of left/right symmetry

• Intrahepatic bile duct development

• Negative regulation of BMP signaling pathway

RARGB is expressed in multiple structures, including blastoderm, head, neural crest, pectoral fin, and tail bud. It is orthologous to human RARG (retinoic acid receptor gamma) . Like other retinoic acid receptors, it functions as a heterodimer with retinoid X receptors (RXRs) and mediates cellular signaling in embryonic morphogenesis, cell growth, and differentiation .

How do RARGB antibodies differ from other RAR subtype antibodies?

RARGB antibodies are specifically designed to detect the gamma B subtype of retinoic acid receptors. This specificity is critical because:

• There are three distinct RAR subtypes (alpha, beta, gamma) with different tissue distribution patterns

• RARalpha is present in most tissue types, while RARbeta and RARgamma have more selective expression patterns

• High-quality RARGB antibodies should not cross-react with other RAR subtypes

For example, the RARγ1 (D3A4) XP® Rabbit mAb recognizes endogenous levels of total RARγ1 protein and does not cross-react with either RARα or RARβ . When selecting an RARGB antibody, researchers should verify that specificity testing has been performed against other RAR family members to ensure target specificity.

What validation steps should be performed before using a new RARGB antibody in critical experiments?

Proper validation of RARGB antibodies is essential for research reproducibility. A systematic validation approach should include:

• Positive and negative control tissues/cells: Use samples known to express or lack RARGB. For RARγ antibodies, positive controls might include lung tissue (mouse) or kidney tissue (rat) .

• Knockout/knockdown verification: If possible, test the antibody in RARGB knockout or knockdown models to confirm specificity.

• Cross-reactivity testing: Verify the antibody doesn't detect other RAR subtypes (alpha and beta).

• Multiple detection methods: Validate using at least two independent methods (e.g., Western blot and immunohistochemistry).

• Peptide competition: Test if the signal can be blocked by pre-incubation with the immunizing peptide.

As highlighted in a UKRN webinar on antibody reproducibility, validation is critical: "Antibodies are known to be an important driver of irreproducibility in research, with issues around the quality of the reagents, the validation of the reagents for the specific purpose, variation in batches and the transparency of reporting"5.

What are the optimal experimental conditions for Western blotting with RARGB antibodies?

For optimal Western blot results with RARGB antibodies, consider the following protocol recommendations:

For difficult-to-detect targets, engineered antibodies with Fc modifications have demonstrated approximately two-fold sensitivity enhancement over wild-type antibodies in Western blot applications .

What are the recommended protocols for immunohistochemistry/immunofluorescence with RARGB antibodies?

For successful immunohistochemistry (IHC) or immunofluorescence (IF) experiments:

• Tissue preparation:

  • For paraffin sections: Use standard formalin fixation and paraffin embedding

  • For frozen sections: Flash freeze in OCT compound

• Antigen retrieval:

  • Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Optimization of retrieval time may be necessary (typically 10-20 minutes)

• Antibody dilution:

  • For IHC: Typically 1:200-1:800 for RARγ antibodies

  • For IF: 1:400-1:800 is recommended

• Incubation conditions:

  • Primary antibody: Overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody: 1 hour at room temperature

• Detection systems:

  • For IHC: DAB or AEC chromogen

  • For IF: Appropriate fluorophore-conjugated secondary antibodies

Some recombinant antibodies have shown exceptional sensitivity and excellent signal-to-noise ratios across different immunoassays, allowing for the study of low-abundant targets .

What are the most common causes of false negatives when using RARGB antibodies, and how can they be addressed?

False negatives with RARGB antibodies can occur for several reasons:

• Inadequate antigen retrieval: Nuclear receptors like RARGB may require more aggressive antigen retrieval

  • Solution: Optimize antigen retrieval methods, try different buffers (citrate vs. EDTA) and extended retrieval times

• Protein degradation: RARs can be susceptible to proteolytic degradation

  • Solution: Use fresh samples, add protease inhibitors, reduce sample processing time

• Insufficient sensitivity: RARGB may be expressed at low levels

  • Solution: Use signal amplification systems, consider engineered antibodies with enhanced sensitivity , or try more sensitive detection methods like CLIA, which has shown higher sensitivity (93.11%) compared to ELISA (82.92%) and LFIA (58.56%) in comparative studies

• Antibody specificity issues: The antibody may not recognize the specific isoform or post-translational modification

  • Solution: Verify which isoforms the antibody detects; for RARG, ensure the antibody can detect RARγ1 or RARγ2 as needed

• Fixation artifacts: Overfixation can mask epitopes

  • Solution: Optimize fixation time or try different fixation methods

If no signal is detected despite optimization, consider validating RARGB expression using an alternative method such as RT-PCR before concluding the protein is not present.

How can researchers address high background or non-specific binding when using RARGB antibodies?

To reduce high background and non-specific binding:

• Optimize blocking:

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (2-3 hours or overnight)

• Antibody concentration:

  • Titrate the antibody to determine optimal concentration

  • For RARγ antibodies, start with recommended dilutions (1:1000 for WB, 1:200-1:800 for IHC) and adjust as needed

• Washing steps:

  • Increase number and duration of washes

  • Use 0.1-0.3% Tween-20 in wash buffers

• Secondary antibody optimization:

  • Use highly cross-adsorbed secondary antibodies

  • Consider secondary antibodies specifically designed for minimal cross-reactivity

• Tissue/sample-specific treatments:

  • For tissues with high endogenous peroxidase activity, use stronger quenching (3% H₂O₂, 10-15 minutes)

  • For tissues with high endogenous biotin, use avidin/biotin blocking kits

• Validation controls:

  • Include isotype controls to identify Fc-mediated binding

  • Use peptide competition assays to confirm specificity

Recent advances in engineered antibodies have demonstrated excellent signal-to-noise ratios across different immunoassays, which may help address background issues .

How can RARGB antibodies be used to study protein-protein interactions in retinoic acid signaling pathways?

RARGB antibodies can reveal protein-protein interactions through several advanced techniques:

• Co-immunoprecipitation (Co-IP):

  • Use RARGB antibodies to pull down the protein complex

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Recommended antibody dilution for IP: 1:50-1:100

• Proximity Ligation Assay (PLA):

  • Detect in situ protein interactions with single-molecule sensitivity

  • Requires two antibodies (anti-RARGB and antibody against suspected interaction partner)

  • Particularly useful for detecting RARγ interactions with RXRs or co-regulators

• Chromatin Immunoprecipitation (ChIP):

  • Map RARGB binding sites on DNA

  • Can be combined with sequencing (ChIP-seq) for genome-wide analysis

  • Critical for understanding RARGB's role as a transcriptional regulator

• FRET/BRET analysis:

  • Detect protein interactions in living cells

  • Requires fusion proteins or labeled antibodies

• Mass spectrometry-based interactomics:

  • Similar to the antigen-specific Fab profiling approach described for autoantibodies

  • Can reveal the entire interaction network

When designing these experiments, consider that "Retinoic acid receptors (RARs) act as heterodimers with retinoid X receptors (RXRs). The RXR/RAR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites" . These interactions are essential for understanding RARGB function.

What are the most reliable approaches for quantifying RARGB expression levels across different tissue samples?

For accurate quantification of RARGB across tissue samples:

• Quantitative immunohistochemistry:

  • Use automated staining platforms for consistency

  • Employ digital image analysis software for objective quantification

  • Include calibration standards on each slide

  • Compare results to standard curves generated with recombinant proteins

• Multiplexed protein assays:

  • Techniques like Luminex or Meso Scale Discovery platforms

  • Allow simultaneous quantification of multiple proteins

  • Provide higher throughput than traditional Western blots

• Enzyme-linked immunosorbent assay (ELISA):

  • Develop sandwich ELISA with capture and detection antibodies

  • Provides highly quantitative results

  • ELISA has shown good sensitivity (82.92%) for protein detection

• Chemiluminescence immunoassay (CLIA):

  • Offers higher sensitivity than ELISA (93.11% vs. 82.92%)

  • Excellent for low-abundance proteins like some nuclear receptors

• Capillary electrophoresis-based protein analysis:

  • Systems like Jess or Wes (ProteinSimple)

  • Provide quantitative Western blot-like data with higher reproducibility

When comparing RARGB levels across samples, normalization to housekeeping proteins and inclusion of common reference samples across experiments are essential for accurate relative quantification.

What novel approaches exist for studying RARGB role in developmental processes using antibodies?

Innovative approaches for studying RARGB in development include:

• Spatial transcriptomics combined with immunostaining:

  • Map RARGB protein expression against transcriptome data

  • Reveals spatial context of RARGB activity in developmental structures

• Intravital microscopy with fluorescently labeled antibodies:

  • Real-time visualization of RARGB in living organisms

  • Particularly useful for zebrafish embryos where rargb plays roles in "determination of left/right symmetry, intrahepatic bile duct development, and negative regulation of BMP signaling pathway"

• Single-cell proteomics with RARGB antibodies:

  • Identify cell populations expressing RARGB during development

  • Can be combined with other markers to create detailed cellular atlases

• CRISPR-engineered reporter systems combined with antibody validation:

  • Generate knock-in fluorescent tags on endogenous RARGB

  • Use antibodies to validate reporter accuracy

• Organ-on-chip models with immunostaining:

  • Study RARGB function in microengineered tissue models

  • Apply antibodies to interrogate signaling dynamics

• Patient-derived organoids:

  • Investigate RARGB in human development using 3D culture systems

  • Particularly relevant for studying congenital disorders linked to retinoic acid signaling

These approaches leverage the specificity of RARGB antibodies while employing cutting-edge technologies to gain deeper insights into developmental processes.

What are the key factors affecting batch-to-batch variability in RARGB antibodies and how can researchers address them?

Batch-to-batch variability is a significant concern in antibody research. For RARGB antibodies:

• Sources of variability:

  • Production method (animal immunization vs. recombinant)

  • Purification procedures

  • Storage conditions

  • Lot-specific characteristics

• Mitigation strategies:

  • Use recombinant antibodies: "Recombinant antibodies offer superior lot-to-lot consistency, continuous supply, and animal-free manufacturing"

  • Perform lot-specific validation: Test each new lot against previous lots using standardized protocols

  • Reserve critical lots: Purchase larger quantities of well-validated lots for long-term studies

  • Implement internal controls: Include reference samples in each experiment to normalize between batches

• Documentation practices:

  • Record lot numbers, validation data, and experimental conditions

  • Use electronic lab notebooks to track antibody performance

  • Share data through antibody validation repositories

As noted in antibody reproducibility discussions, "issues around the quality of the reagents, the validation of the reagents for the specific purpose, variation in batches and the transparency of reporting of both methods and results" are key drivers of irreproducibility5.

How do different antibody generation technologies impact RARGB antibody quality and experimental outcomes?

Different antibody generation technologies have distinct impacts on RARGB antibody quality:

For example, phage display-derived monoclonal antibodies binding to RGMb demonstrated "high affinities of 1.4 nM and 0.72 nM" with potent inhibition of specific protein interactions , showing how advanced generation technologies can yield highly specific research tools.

When selecting an RARGB antibody, consider the technology's impact on your specific research question and experimental design.

How are technological advances in antibody engineering changing the landscape of RARGB research?

Recent technological advances are revolutionizing RARGB research through:

• Fc-engineered antibodies:

  • "Using proprietary technology, we have engineered the Fc region of the rabbit recombinant monoclonal antibody"

  • These modifications enhance sensitivity across multiple applications

  • Allow detection of lower abundance targets without changing experimental workflows

• AI-assisted epitope analysis:

  • Systems like "MODELAGON™ (AI-assisted epitope analysis system)" help identify optimal immunogenic regions

  • Enhances antibody design for challenging nuclear receptors like RARGB

  • Incorporates 3D modeling for accurate selection of functional epitopes

• Multiparametric antibodies:

  • Dual-labeled antibodies allow simultaneous detection of location and activation state

  • Important for understanding RARGB's role in dynamic cellular processes

• Single-domain antibodies and nanobodies:

  • Smaller binding molecules with enhanced tissue penetration

  • Valuable for detecting RARGB in complex tissue structures

• Structure-guided antibody development:

  • X-ray crystallography and cryo-EM of antibody-antigen complexes

  • Similar to approaches used for virus-targeting antibodies: "X-ray structure of one of the most potent and most broadly reactive human bnAbs"

  • Reveals binding determinants that can guide next-generation antibody development

These advances expand the toolkit available for RARGB research, enabling more precise interrogation of its biological functions and pathway interactions.

What are the most promising applications of RARGB antibodies in translational research connecting basic science to clinical implications?

Translational applications of RARGB antibodies include:

• Cancer research:

  • RARGB expression as a potential biomarker

  • RARγ is "the predominant subtype in human and mouse epidermis, representing 90% of the RARs in this tissue"

  • Antibodies enable study of dysregulation in carcinogenesis

• Developmental disorders:

  • Investigation of RARGB's role in congenital abnormalities

  • Particularly relevant for disorders involving "determination of left/right symmetry" and "intrahepatic bile duct development"

• Regenerative medicine:

  • Monitoring RARGB expression during stem cell differentiation

  • RARγ plays a role in "hematopoietic stem cell maintenance"

• Drug discovery:

  • Screening compounds that modulate RARGB activity

  • Antibodies provide critical tools for target engagement studies

• Personalized medicine:

  • Potential development of companion diagnostics based on RARGB expression

  • Similar to approaches using "antibody serology tests" in other fields

Through these applications, RARGB antibodies connect basic science insights to potential clinical innovations, particularly in fields where retinoic acid signaling plays important regulatory roles.