RXRB Antibody

What is RXRB and what is its biological significance?

RXRB (Retinoid X Receptor beta) is a member of the nuclear receptor superfamily that binds selectively and with high affinity to the vitamin A derivative, 9-cis-retinoic acid . It functions as a type-II nuclear hormone receptor that localizes to the nuclear compartment independent of ligand binding . RXRB forms heterodimers with nuclear hormone receptor subfamily 1 proteins, including thyroid hormone receptor, retinoic acid receptors, vitamin D receptor, peroxisome proliferator-activated receptors, liver X receptors, and farnesoid X receptor . Through these interactions, RXRB regulates a wide variety of genes important for metabolic processes .

The RXRB gene is located within the major histocompatibility complex (MHC) class II region on chromosome 6 . Structurally, RXRB possesses a characteristic tripartite modular structure consisting of a highly conserved central region containing the C4/C5 zinc-finger domain responsible for DNA binding, and a relatively well-conserved C-terminal region containing the hormone binding and dimerization domains .

What types of RXRB antibodies are available for research applications?

Several types of RXRB antibodies are available for various research applications:

This diversity allows researchers to select the most appropriate antibody based on their specific application, target species, and experimental design .

How should researchers select the appropriate RXRB antibody for their experimental needs?

When selecting an RXRB antibody, researchers should consider several key factors:

• Application compatibility: Different antibodies are optimized for specific applications like Western blotting (WB), immunoprecipitation (IP), immunohistochemistry (IHC), or flow cytometry .

• Species reactivity: Verify that the antibody has been validated in your species of interest. For instance, some antibodies are only validated for human and mouse samples, while others may have predicted reactivity based on sequence homology that requires experimental confirmation .

• Clonality: Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes and may provide higher sensitivity but potentially more background .

• Validation data: Review supporting data showing the antibody's performance in relevant applications. For example, Western blot results showing the expected molecular weight (typically 55-72 kDa for RXRB depending on the source) .

• Formulation requirements: Consider whether your experiment requires a specific formulation (e.g., BSA-free, azide-free) .

How can researchers validate the specificity of RXRB antibodies?

Validating antibody specificity is critical for obtaining reliable results. Although the search results don't provide a specific protocol for RXRB antibody validation, we can adapt approaches used for similar nuclear receptors:

• Western blot validation: Confirm a single specific band at the predicted molecular weight (typically 55-72 kDa for RXRB) . Multiple bands may indicate degradation, isoforms, or non-specific binding.

• Transfection studies: Compare antibody reactivity in cells transiently expressing RXRB versus non-transfected controls. For example, one antibody showed a ~60 kDa band in RXRB-transfected HEK293 cells that was absent in non-transfected cells .

• Cross-species validation: If working across species, verify antibody performance despite predicted reactivity based on sequence homology .

• Positive and negative controls: Include tissues or cell lines known to express or lack RXRB expression.

• Knockdown/knockout validation: If possible, compare staining in wild-type versus RXRB-depleted samples to confirm specificity.

What is the optimal protocol for Western blotting with RXRB antibodies?

Based on the available data, the following Western blotting parameters are recommended:

• Sample preparation: Prepare cell or tissue lysates using standard protocols.

• Expected molecular weight: Prepare to detect bands between 55-72 kDa depending on the antibody and sample type .

• Antibody dilution: Typically use primary antibody at 1:1000 dilution .

• Secondary antibody: Use appropriate species-specific HRP-conjugated secondary antibody (e.g., Goat Anti-Rabbit IgG H&L (HRP) at 1:5000 dilution) .

• Exposure time: Approximately 3 minutes, adjusting based on signal strength .

A specific example from the literature shows:

• Primary antibody: Anti-RXRB antibody (ab5793) at 1/1000 dilution

• Control protein: Recombinant Human RXRB protein (ab82122) at 0.1 µg

• Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

• Predicted band size: 57 kDa

• Exposure time: 3 minutes

How can researchers optimize immunohistochemistry protocols for RXRB detection?

While specific RXRB immunohistochemistry protocols weren't detailed in the search results, we can adapt approaches from related nuclear receptor studies:

• Antibody validation: First verify antibody specificity via Western blot before proceeding to IHC .

• Section preparation: Use 4 µm thick sections from either full-face tissue or tissue microarrays .

• Detection system: Employ a sensitive detection system (e.g., polymer-based systems) .

• Antibody dilution and incubation: Optimize through titration; related nuclear receptor antibodies have been used at 1:300 dilution with 24-hour incubation .

• Quantification method: Consider using the modified Histo-score (H-score) method, which accounts for both staining intensity and percentage positivity .

• Digital imaging: High-resolution digital imaging can facilitate consistent scoring of tissue microarray cores .

• Multiple scoring: Implement blind double-scoring by at least two researchers to ensure reliability (aim for intra-class correlation coefficient >0.8) .

What experimental approaches can researchers use to study RXRB binding dynamics?

Based on studies of RAR/RXR binding dynamics, the following approaches can be adapted for RXRB research:

• Chromatin Immunoprecipitation sequencing (ChIP-seq): This technique enables genome-wide identification of RXRB binding sites and temporal analysis of binding events during biological processes .

• Statistical filtering: Apply rigorous statistical tests (e.g., Poisson Margin Test) to detect regions with significant differences between RXRB binding and control signals, using appropriate adjustment for multiple testing (e.g., Benjamini-Hochberg adjustment) .

• Motif analysis: Use bioinformatic tools like MEME Suite for de novo motif discovery in RXRB binding regions .

• Dynamic occupancy analysis: Analyze the proportion of genomic regions showing different binding profiles (bound/unbound) across various experimental conditions or time points .

• Co-occupancy analysis: Determine regions simultaneously bound by RXRB and its heterodimeric partners to understand cooperative binding events .

How can researchers investigate RXRB heterodimerization with other nuclear receptors?

RXRB forms heterodimers with multiple nuclear receptors to regulate gene expression. Several approaches can be used to study these interactions:

• Co-immunoprecipitation (Co-IP): Use RXRB antibodies to immunoprecipitate protein complexes, followed by Western blotting to detect associated nuclear receptors. The RXRβ antibody #8715 has been validated for immunoprecipitation at 1:50 dilution .

• Sequential ChIP (Re-ChIP): Perform consecutive immunoprecipitations with antibodies against RXRB and potential partners to identify genomic regions where both proteins bind together.

• ChIP-seq comparative analysis: Compare binding profiles of RXRB and other nuclear receptors to identify regions of co-occupancy. As noted in RAR/RXR studies, "66% of the regions interacting with the heterodimeric complex in absence of RA remain bound over time" .

• Differential binding analysis: Analyze how heterodimerization changes under different conditions. For example, RAR/RXR studies showed that "upon RA stimulation, the number of RAR/RXR bound regions increases massively but transiently" .

• Negativity threshold definition: Establish appropriate thresholds to identify independent binding events, as RAR/RXR studies found that "18% of all RAR/RXR binding regions are occupied by RXR but not by RAR in untreated F9 cells" .

What are the challenges in studying cross-species conservation of RXRB function?

When investigating RXRB across different species, researchers should consider:

• Sequence homology vs. experimental validation: While antibody reactivity can be predicted based on sequence homology, experimental validation is essential. As noted, "The antigen sequence used to produce this antibody shares 100% sequence homology with the species listed here, but reactivity has not been tested or confirmed to work by CST" .

• Molecular weight variations: RXRB may exhibit different molecular weights across species due to post-translational modifications or species-specific isoforms .

• Functional conservation assessment: Despite sequence conservation, RXRB's functional interactions with other nuclear receptors may vary between species, requiring careful interpretation of cross-species data.

• Species-specific expression patterns: RXRB expression levels and tissue distribution might differ between species, necessitating baseline characterization for each species.

• Warranty considerations: Commercial antibodies may not be covered by performance guarantees when used with species not specifically validated by the manufacturer .

How can RXRB antibodies be utilized in cancer research applications?

While the search results don't specifically address RXRB in cancer, related nuclear receptor research suggests several applications:

• Expression profiling: RXRB antibodies can be used for immunohistochemical assessment of RXRB expression in tumor samples, potentially serving as prognostic or predictive biomarkers .

• Binding partner analysis: Investigating how RXRB heterodimerization patterns change in cancer cells may reveal altered signaling mechanisms.

• Therapeutic target assessment: As nuclear receptors are druggable targets, understanding RXRB expression and activity in cancer cells could inform therapeutic strategies using retinoids or other nuclear receptor ligands.

• Mechanistic studies: RXRB antibodies can be employed in studies investigating the role of RXRB in cancer cell proliferation, apoptosis, and differentiation through various functional assays.

Why might Western blots with RXRB antibodies show unexpected banding patterns?

When Western blots with RXRB antibodies show unexpected bands, consider these possible explanations:

• Variable molecular weights: RXRB has been reported at different molecular weights:

  • 70-72 kDa in human and mouse samples

  • 57 kDa predicted band size in human samples

  • ~55 kDa observed in A431 cell lysates

  • ~60 kDa in RXRB-transfected HEK293 cells

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter RXRB's apparent molecular weight.

• Isoforms: Alternative splicing may generate different RXRB isoforms with varying molecular weights.

• Degradation products: Sample handling or storage issues may cause protein degradation, resulting in lower molecular weight bands.

• Cross-reactivity: The antibody may detect related proteins such as RXRα or RXRγ due to sequence homology.

• Non-specific binding: Some antibodies may bind to unrelated proteins, especially at suboptimal dilutions.

To address these issues, researchers should optimize sample preparation, validate with appropriate controls, and consider using multiple antibodies targeting different RXRB epitopes.

What are the critical parameters for successful immunoprecipitation of RXRB?

Based on available information about RXRB immunoprecipitation:

• Antibody selection: Choose an antibody validated specifically for immunoprecipitation, such as RXRβ Antibody #8715, which is recommended at 1:50 dilution for IP applications .

• Lysate preparation: Optimize lysis conditions to efficiently extract nuclear proteins while preserving protein-protein interactions relevant to RXRB function.

• Controls: Include appropriate negative controls (non-specific IgG, lysates from cells not expressing RXRB) to distinguish specific from non-specific interactions.

• Detection strategy: For Western blot detection after IP, consider using a different RXRB antibody that recognizes a different epitope to confirm specificity.

• Co-IP considerations: When studying RXRB interactions with other nuclear receptors, optimize conditions to preserve these interactions, which may be sensitive to detergent type and concentration.

• Cross-linking options: For transient or weak interactions, consider using cross-linking approaches to stabilize protein complexes before immunoprecipitation.

How can researchers address inconsistent results when comparing different RXRB antibodies?

When faced with discrepancies between different RXRB antibodies, consider:

• Epitope differences: Different antibodies may target distinct regions of RXRB, potentially affecting detection of specific isoforms or modified forms of the protein.

• Application-specific optimization: An antibody performing well in Western blotting may not be optimal for IHC or IP applications. Each antibody should be validated for the specific application .

• Lot-to-lot variation: Consider testing different lots of the same antibody or requesting data on lot-to-lot consistency from manufacturers.

• Protocol optimization: Each antibody may require specific conditions regarding dilution, incubation time, temperature, and detection systems.

• Sample preparation impact: Different sample preparation methods may expose or mask epitopes differentially across antibodies.

• Independent validation methods: Supplement antibody-based detection with orthogonal approaches (mRNA analysis, overexpression/knockdown studies) to corroborate findings.

• Control experiments: Perform side-by-side comparisons with appropriate positive and negative controls for each antibody.

What emerging technologies might enhance RXRB antibody applications?

While not explicitly mentioned in the search results, several emerging technologies could enhance RXRB research:

• Single-cell analysis: Combining RXRB antibodies with single-cell technologies could reveal cell-to-cell variations in RXRB expression and heterodimer formation within heterogeneous tissues.

• Proximity labeling methods: BioID or APEX2-based approaches using RXRB fusions could identify novel interaction partners in living cells.

• Super-resolution microscopy: Advanced imaging techniques could provide insights into the subnuclear localization of RXRB and its colocalization with other nuclear receptors at unprecedented resolution.

• CRISPR-based approaches: Endogenous tagging of RXRB could enable visualization and purification of RXRB complexes without overexpression artifacts.

• Integrative multi-omics: Combining RXRB ChIP-seq with transcriptomics, proteomics, and metabolomics could provide comprehensive understanding of RXRB-dependent regulatory networks.

How might RXRB research contribute to therapeutic developments?

The nuclear receptor superfamily represents important therapeutic targets, suggesting several potential applications for RXRB research:

• Retinoid-based therapies: Understanding RXRB's role in mediating retinoid effects could inform the development of more selective retinoid therapies with fewer side effects.

• Nuclear receptor modulators: Insights into RXRB heterodimerization could guide the development of compounds that selectively modulate specific RXRB-containing complexes.

• Biomarker development: RXRB expression or localization patterns might serve as biomarkers for disease states or treatment responses, particularly in contexts where nuclear receptor signaling is dysregulated.

• Precision medicine approaches: Characterizing individual variations in RXRB expression or function could help stratify patients for targeted therapies directed at nuclear receptor pathways.

• Gene therapy strategies: RXRB-based gene regulatory systems could potentially be engineered for therapeutic gene expression control in various disease contexts.