rarga Antibody

What is RARG and why are antibodies against it important in research?

RARG (Retinoic Acid Receptor Gamma) is a nuclear hormone receptor that functions as a ligand-dependent transcriptional regulator. It belongs to the steroid and thyroid hormone superfamily of nuclear receptor proteins which exert their effects by binding to specific DNA response elements, thus regulating gene expression in target cells . RARG plays critical roles in cell differentiation, proliferation, and apoptosis, and has been implicated in various diseases including cancer and developmental disorders .

Antibodies against RARG are crucial research tools because:

• They enable detection and analysis of RARG expression patterns across different tissues

• They facilitate investigation of RARG's role in normal development and disease states

• They allow researchers to study protein-protein interactions involving RARG

• They can be used to examine the relationship between RARG expression and clinical outcomes

For instance, studies have demonstrated that RARG is involved in multiple signaling pathways relevant to cancer progression, making RARG antibodies valuable tools in oncology research .

How do I select between monoclonal and polyclonal RARG antibodies for my experiments?

The choice between monoclonal and polyclonal RARG antibodies depends on your specific research needs:

Monoclonal RARG Antibodies:

• Offer high specificity for a single epitope

• Provide consistent lot-to-lot reproducibility

• Ideal for detecting specific RARG isoforms

• Excellent for quantitative applications

• Better for distinguishing between closely related RAR family members

Polyclonal RARG Antibodies:

• Recognize multiple epitopes, potentially increasing signal strength

• More tolerant to minor protein denaturation or conformational changes

• Can detect RARG across different species more effectively

• May be more suitable for applications like immunoprecipitation

For example, the monoclonal antibody described in search result offers high specificity for human, mouse, and rat RARG, making it ideal for comparative studies across species. In contrast, polyclonal antibodies like CAB7448 target a specific sequence (amino acids 168-199) of human RARG, providing recognition of multiple epitopes within that region.

What validation methods should I use to confirm RARG antibody specificity?

Thorough validation of RARG antibodies is critical for reliable results. The following validation methods are recommended based on established practices in the field:

• Western blot analysis:

  • The antibody should detect a predominant band at 48-55 kDa (the expected molecular weight of RARG)

  • Include positive controls (cell lines known to express RARG, such as MCF-7, HeLa)

  • Include negative controls (samples with low or no RARG expression)

  • Consider using RARG knockout or knockdown samples as definitive negative controls

• Peptide competition assay:

  • Pre-incubation of the antibody with the immunizing peptide should block recognition of RARG

  • Use unrelated peptides as negative controls

• Cross-reactivity testing:

  • Test against other RAR family members (RARα, RARβ) to ensure specificity

  • As demonstrated in result , a well-validated antibody should detect RARG but not other RAR forms

• Multiple antibody concordance:

  • Compare results with other validated RARG antibodies targeting different epitopes

  • Consistent results across different antibodies increase confidence in specificity

• Immunoprecipitation-Mass Spectrometry:

  • For definitive validation, immunoprecipitate with the RARG antibody and confirm identity by mass spectrometry

For example, the validation process described in result demonstrates how a rigorously validated antibody shows a single band (or specific multiple bands for protein isoforms) of correct molecular size with known positive and negative controls, and equivalent performance under various assay conditions.

What applications can RARG antibodies be successfully used for?

RARG antibodies have been validated for multiple applications, each requiring specific optimization:

Western Blot (WB):

• Most commonly used application with recommended dilutions typically between 1:500-1:1000

• Can detect RARG at approximately 48-55 kDa

• Useful for quantifying expression levels in different tissues or experimental conditions

Immunohistochemistry (IHC):

• Enables localization of RARG in tissue sections

• Can reveal expression patterns across different cell types within tissues

• Critical for understanding RARG distribution in normal and pathological conditions

Immunofluorescence (IF):

• Allows subcellular localization studies

• Can be combined with other markers for co-localization studies

• Useful for studying nuclear translocation in response to ligands

Immunoprecipitation (IP):

• Facilitates study of protein-protein interactions

• Recommended usage: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Essential for identifying RARG binding partners

Chromatin Immunoprecipitation (ChIP):

• Crucial for studying RARG binding to DNA and gene regulation

• Has been successfully used to identify RARG target genes

Gel Shift/EMSA (Electrophoretic Mobility Shift Assay):

• Used to study RARG binding to specific DNA sequences

• Can demonstrate RARG-RXR heterodimer interactions with response elements

For example, PA3-813 antibody has been successfully used in Western blot, immunoprecipitation, and gel shift procedures, demonstrating efficient super shifting of the human RARG-1/retinoid X receptor (RXR)/RAR response element complex in gel super shift experiments .

How do RARG antibodies differ in their recognition of RARG isoforms?

RARG exists in multiple isoforms, primarily RARG-1 and RARG-2, which differ in their N-terminal regions. Selecting antibodies that can distinguish between these isoforms is crucial for specific research questions:

Isoform-specific antibodies:

• Some antibodies, like PA3-813, are specifically designed to detect RARG-1 but not RARG-2

• These antibodies typically target the unique N-terminal region of RARG-1

• Essential for studies investigating isoform-specific functions

Pan-RARG antibodies:

• Target conserved regions present in all RARG isoforms

• Useful for detecting total RARG expression regardless of isoform

• Typically target the DNA-binding domain or ligand-binding domain

Research considerations:

• RARG-1 is predominantly expressed in skin

• RARG-2 is expressed early in embryogenesis and in embryonal carcinoma cells

• Different isoforms may have distinct biological functions and tissue distributions

For example, PA3-813 immunizing peptide corresponds to the N-terminus of RARG-1, enabling specific detection of this isoform without cross-reactivity with RARG-2 . Understanding which isoform your antibody detects is critical for interpreting experimental results correctly.

How can RARG antibodies be used to investigate RARG's role in cancer progression?

RARG antibodies have become instrumental in elucidating RARG's role in cancer development and progression. Several methodological approaches have proven effective:

Expression analysis in cancer tissues:

• IHC with RARG antibodies can reveal expression patterns across different cancer types and stages

• Studies have demonstrated that high RARG expression correlates with accelerated ovarian cancer progression

• Quantitative Western blot analysis can measure RARG levels across patient samples and correlate with clinical outcomes

Functional studies:

• Combine RARG antibodies with knockdown/knockout approaches to validate specificity

• In studies where RARG expression was downregulated, significant suppression of proliferation and colony formation capacity of cancer cells was observed

• RARG antibodies can confirm successful knockdown at the protein level

Mechanistic investigations:

• ChIP assays using RARG antibodies can identify cancer-relevant target genes

• Co-immunoprecipitation with RARG antibodies can reveal cancer-specific protein interactions

• Immunofluorescence can track RARG subcellular localization in response to cancer therapies

In vivo xenograft models:

• RARG antibodies can validate RARG expression in xenograft tumors

• IHC on tumor tissues can correlate RARG expression with proliferation markers like Ki-67 and PCNA

For example, research has demonstrated that downregulation of RARG expression significantly suppressed the proliferation and colony formation capacity of ovarian cancer cells, and RARG antibodies were essential for validating these findings through Western blot analysis .

What approaches should I use to study RARG-RXR heterodimer complexes with antibodies?

RARG functions primarily by forming heterodimers with Retinoid X Receptors (RXRs) to regulate gene expression. Studying these complexes requires specialized approaches:

Co-immunoprecipitation (Co-IP):

• Use RARG antibodies to immunoprecipitate the complex, then detect RXR with specific antibodies

• Alternatively, immunoprecipitate with RXR antibodies and detect RARG

• Include appropriate controls (IgG, lysate input)

• Consider crosslinking to stabilize transient interactions

Electrophoretic Mobility Shift Assay (EMSA) with supershift:

• RARG antibodies can be used to "supershift" RARG-RXR-DNA complexes

• PA3-813 has been shown to efficiently supershift the human RARG-1/RXR/RAR response element complex

• This approach confirms the presence of RARG in DNA-binding complexes

Proximity Ligation Assay (PLA):

• Combines RARG and RXR antibodies to visualize interactions in situ

• Generates fluorescent signals only when the proteins are in close proximity

• Provides spatial information about where heterodimers form within cells

Chromatin Immunoprecipitation (ChIP):

• Sequential ChIP (re-ChIP) using first RARG antibodies then RXR antibodies

• Identifies genomic loci bound by RARG-RXR heterodimers specifically

• Compare with single ChIP results to distinguish heterodimer-specific binding sites

For heterodimer studies, it's important to note that "Collins et al. developed a HL60 cell line resistant to differentiation by ATRA. This cell line harbored a dominant negative mutant RARα... Differentiation of these cells under the influence of ATRA was restored by infection with a retrovirus expressing wild-type RARα, RARβ, or RARγ" , demonstrating the functional redundancy and importance of RARG-RXR signaling.

How can I use RARG antibodies effectively in ChIP-seq experiments to study RARG-mediated gene regulation?

ChIP-seq with RARG antibodies is a powerful approach to identify direct genomic targets of RARG. The following methodological considerations should be addressed:

Antibody selection for ChIP:

• Use antibodies specifically validated for ChIP applications

• Look for antibodies with demonstrated success in published ChIP studies

• Consider monoclonal antibodies for higher specificity and reproducibility

Optimization of ChIP protocol:

• Perform antibody titration to determine optimal concentration

• Typically, 0.5-5 μg antibody per ChIP reaction is recommended

• Include appropriate positive controls (known RARG target genes) and negative controls (IgG, genomic regions not bound by RARG)

Cross-linking and sonication:

• Optimize formaldehyde cross-linking time (typically 10-15 minutes)

• Ensure proper chromatin fragmentation (200-500 bp fragments)

• Verify sonication efficiency by agarose gel electrophoresis

Data analysis considerations:

• Use appropriate peak calling algorithms (MACS2, etc.)

• Perform motif enrichment analysis to identify RARG binding motifs

• Integrate with RNA-seq data to correlate binding with transcriptional changes

Validation of ChIP-seq results:

• Confirm selected binding sites by ChIP-qPCR

• Perform reporter assays with identified response elements

• Consider the possibility of indirect binding through protein-protein interactions

For example, search result indicates that RARG antibodies have been successfully used in ChIP applications, with multiple publications citing their use. When interpreting ChIP-seq data, remember that RARG can bind DNA through various mechanisms, including direct binding to retinoic acid response elements (RAREs) as a heterodimer with RXR.

What methodological approaches can address cross-reactivity between RARG antibodies and other RAR family members?

Cross-reactivity with other RAR family members (RARα and RARβ) is a common challenge when working with RARG antibodies. These approaches can help address this issue:

Comprehensive validation strategies:

• Test antibodies against recombinant RARα, RARβ, and RARG proteins

• Perform Western blots on cells overexpressing each RAR family member

• Include knockout/knockdown samples for each RAR family member as definitive controls

Epitope selection for high specificity:

• Choose antibodies targeting regions with low sequence homology between RAR family members

• N-terminal regions often show greater sequence divergence among RAR subtypes

• As demonstrated in result , PA3-813 detects RARG-1 but not RARα or RARβ due to its N-terminal epitope

Competitive blocking experiments:

• Pre-incubate antibodies with peptides from RARα, RARβ, and RARG

• Only the peptide corresponding to the true target should block antibody binding

• For example, "preincubation of the antibodies with the synthetic RAR alpha peptide, but not with the RAR beta or RAR gamma peptides, blocked recognition of the approximately 55 kDa RAR alpha protein on western blots"

Alternative confirmation approaches:

• Use multiple antibodies targeting different epitopes of RARG

• Combine antibody-based detection with other methods (e.g., mass spectrometry)

• Consider using RNA-based methods (RT-PCR, RNA-seq) to complement protein detection

Cross-validation with functional assays:

• Correlate antibody staining with known functional differences between RAR family members

• For instance, "RARα expression in the resistant cells restored myeloid differentiation, suggesting that the mutant receptor may have acted in a dominant negative mode"

How can computational and experimental approaches complement each other in designing highly specific RARG antibodies?

The integration of computational methods with experimental validation represents a cutting-edge approach to developing highly specific RARG antibodies:

Computational epitope prediction:

• In silico analysis can identify regions of RARG with minimal homology to other proteins

• Structure-based approaches can predict surface-exposed regions unique to RARG

• Machine learning algorithms can help predict antigenic determinants with high immunogenicity

Binding mode analysis:

• Computational models can predict different binding modes between antibodies and RARG

• "Our biophysics-informed model is trained on a set of experimentally selected antibodies and associates to each potential ligand a distinct binding mode, which enables the prediction and generation of specific variants beyond those observed in the experiments"

• These models can help design antibodies with customized specificity profiles

Experimental validation pipeline:

• Start with phage display to select antibodies against RARG

• Use high-throughput sequencing to analyze selected antibodies

• Employ computational analysis to identify potentially cross-reactive clones

• Experimentally validate promising candidates

Iterative refinement:

• Computational analysis of experimental data can guide antibody engineering

• "Using data from phage display experiments, we show that the model successfully disentangles these modes, even when they are associated with chemically very similar ligands"

• This approach can generate antibodies with custom specificity profiles not present in initial libraries

As demonstrated in result , combining experimental selection with computational analysis allows researchers to "demonstrate and validate experimentally the computational design of antibodies with customized specificity profiles, either with specific high affinity for a particular target ligand, or with cross-specificity for multiple target ligands."

What are the best methodological approaches for using RARG antibodies in conjunction with ATRA treatment studies?

All-trans retinoic acid (ATRA) is a natural ligand for RARG, and studying their interactions requires specific methodological considerations:

Time-course experiments:

• Monitor RARG expression, localization, and activity at multiple time points after ATRA treatment

• Western blot with RARG antibodies can detect expression changes

• Immunofluorescence can reveal nuclear translocation dynamics

Chromatin occupancy changes:

• ChIP-seq with RARG antibodies before and after ATRA treatment

• Reveals how ligand binding affects genomic targeting

• Can identify ligand-dependent and ligand-independent binding sites

Protein-protein interaction shifts:

• Co-IP with RARG antibodies followed by mass spectrometry

• Identifies how the RARG interactome changes upon ATRA binding

• Critical for understanding context-specific functions

Functional readouts:

• Correlate RARG binding (detected with antibodies) with transcriptional changes

• In myeloid differentiation models, "differentiation of these cells under the influence of ATRA was restored by infection with a retrovirus expressing wild-type RARα, RARβ, or RARγ"

• Include appropriate controls (RARG antagonists, RAR-selective ligands)

Dose-response considerations:

• Test multiple ATRA concentrations to distinguish physiological from pharmacological effects

• Use RARG antibodies to correlate receptor occupancy with downstream responses

• Remember that "RA alone, and in combination with stimuli that are ligands for the Toll-like receptor family, can augment the adaptive immune response"

For studying immune modulation specifically, research has shown that "signals generated by the binding of Ag (signal 1) to its receptors, and costimulatory/accessory molecules (signal 2) to their respective receptors... RA could be an important 'signal 4,' when RA is present at an adequate concentration during the period of initial B-cell and T-cell stimulation" .

How can RARG antibodies be used to investigate differential functions of RARG in various tissues and cell types?

RARG exhibits tissue-specific expression patterns and functions, and antibodies are essential tools for investigating these differences:

Comparative tissue profiling:

• Use RARG antibodies for IHC across multiple tissue types

• For example, "RARG-1 has been found to be predominantly expressed in skin, while RARG-2 has been found early in embryogenesis and in embryonal carcinoma cells"

• Create expression atlases across development or disease progression

Cell type-specific analysis:

• Combine RARG antibodies with cell type-specific markers for co-localization studies

• Flow cytometry with RARG antibodies can quantify expression in specific cell populations

• Single-cell Western blot can reveal heterogeneity within tissues

Functional correlation studies:

• Compare RARG binding patterns (via ChIP-seq) across different cell types

• Correlate RARG occupancy with tissue-specific gene expression

• In cell differentiation models, RARG has demonstrated importance in myeloid differentiation

Knockout/knockdown validation:

• Use RARG antibodies to confirm complete protein loss in tissue-specific knockout models

• Compare phenotypic consequences across different tissues

• For example, in ovarian cancer research, RARG knockdown significantly suppressed proliferation

Subcellular localization differences:

• RARG primarily localizes to the nucleus but may exhibit tissue-specific distribution patterns

• Use fractionation followed by Western blot with RARG antibodies to compare nuclear/cytoplasmic ratios

• Immunofluorescence can reveal subtle differences in subnuclear localization

A comprehensive approach might involve "various applications, including immunohistochemistry, flow cytometry, and Western blotting, allowing for versatile and reliable detection of RARG in different experimental settings" across multiple tissue types to build a complete picture of RARG's tissue-specific functions.

What are the optimal storage conditions and handling procedures for RARG antibodies?

Proper storage and handling of RARG antibodies are critical for maintaining their activity and specificity over time:

Storage conditions:

• Most RARG antibodies should be stored at -20°C

• For example, the storage recommendation for the Proteintech antibody is: "Store at -20°C. Stable for one year after shipment"

• Many antibodies are supplied in storage buffer containing glycerol (typically 50%), which prevents freezing and thawing damage

• Some antibodies contain preservatives like sodium azide (e.g., "PBS with 0.02% sodium azide and 50% glycerol pH 7.3")

Aliquoting recommendations:

• For antibodies without glycerol, aliquoting is essential to avoid freeze-thaw cycles

• For antibodies with glycerol, the manufacturer may state that "Aliquoting is unnecessary for -20°C storage"

• Use sterile microcentrifuge tubes for aliquoting

• Typical aliquot volumes range from 10-20 μL based on experimental needs

Thawing procedure:

• Thaw antibodies on ice or at 4°C

• Avoid multiple freeze-thaw cycles which can lead to denaturation and loss of activity

• Centrifuge briefly after thawing to collect antibody at the bottom of the tube

Working solution preparation:

• Dilute antibodies in appropriate buffer just before use

• For Western blot, BSA-containing buffers often provide better results than milk-based blockers

• Follow manufacturer-recommended dilutions as starting points (typically 1:500-1:1000 for Western blot)

Quality control checks:

• Monitor antibody performance over time

• Include positive controls in each experiment to detect potential degradation

• Consider validated recombinant RARG protein as a standard for quality control

Following these storage and handling procedures will help ensure consistent, reproducible results when working with RARG antibodies.

How do I troubleshoot common issues when using RARG antibodies in experimental applications?

Even with validated RARG antibodies, researchers may encounter technical challenges. Here are methodological approaches to address common issues:

Western Blot: No signal or weak signal

• Increase antibody concentration (try 2-5× the recommended dilution)

• Extend primary antibody incubation (overnight at 4°C)

• Increase protein loading (50-100 μg total protein)

• Verify transfer efficiency with reversible staining

• Use enhanced chemiluminescence (ECL) detection as used in result

Western Blot: Multiple bands

• Determine if bands represent isoforms, degradation products, or non-specific binding

• Use positive controls with known RARG expression

• Consider peptide competition assay to identify specific bands

• Optimize blocking conditions (try different blockers like BSA, milk, commercial blockers)

Immunohistochemistry: High background

• Increase blocking time and concentration

• Try different blocking agents (normal serum matching secondary antibody species)

• Optimize antibody dilution (typically higher dilution than for Western blot)

• Include appropriate negative controls (no primary antibody, isotype control)

Immunoprecipitation: Low yield

• Increase antibody amount (0.5-4.0 μg recommended for IP)

• Extend incubation time with antibody (overnight at 4°C)

• Ensure efficient cell lysis (try different lysis buffers)

• Consider crosslinking antibody to beads to prevent co-elution

ChIP: Poor enrichment

• Optimize cross-linking conditions

• Ensure proper sonication (200-500 bp fragments)

• Increase antibody amount or incubation time

• Include positive control regions (known RARG binding sites)

Flow Cytometry: Poor separation

• Optimize fixation and permeabilization for nuclear antigens like RARG

• Try different permeabilization reagents (Triton X-100, saponin, methanol)

• Increase antibody concentration

• Include fluorescence-minus-one (FMO) controls

For example, result demonstrates successful Western blot detection of RARG using RIPA buffer with protease inhibitors for protein extraction, a 1:1000 antibody dilution, and GAPDH (1:5000) as a loading control, with visualization using a super enhanced chemiluminescence detection reagent.

How can I quantitatively analyze RARG expression using antibodies in complex biological samples?

Quantitative analysis of RARG requires standardized approaches to ensure reproducibility and accuracy:

Western Blot Quantification:

• Use increasing amounts of recombinant RARG protein to create a standard curve

• Normalize RARG band intensity to a loading control (GAPDH, β-actin)

• Employ digital image analysis software with background subtraction

• Report relative expression as fold-change compared to control samples

Flow Cytometry:

• Use calibration beads with known quantities of fluorophores

• Calculate molecules of equivalent soluble fluorochrome (MESF)

• Include samples with known RARG expression levels as internal standards

• Present data as median fluorescence intensity (MFI) or percent positive cells

ELISA:

• Develop sandwich ELISA using two antibodies recognizing different RARG epitopes

• Include recombinant RARG protein standards to generate absolute quantification

• Perform spike-recovery experiments to validate accuracy

• Report results as ng/mL or pg/μg total protein

Mass Spectrometry with Antibody Enrichment:

• Immunoprecipitate RARG from samples using specific antibodies

• Analyze by targeted mass spectrometry with isotope-labeled peptide standards

• Quantify using multiple reaction monitoring (MRM)

• This approach combines antibody specificity with the quantitative power of MS

Reverse Phase Protein Array (RPPA):

• "RPPA is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples"

• Samples are "robotically arrayed as microspots on nitrocellulose-coated glass slides"

• Each slide is probed with RARG antibody

• Provides high sensitivity quantification across many samples simultaneously

When analyzing data, ensure appropriate statistical methods are applied: "The relative cell viability (%) was finally calculated" and "The colonies were counted" demonstrate quantitative approaches used in published RARG research.