vdrb Antibody

What is VDRB and what role does it play in cellular function?

VDRB (Vitamin D Receptor B) is a nuclear receptor for calcitriol, the active form of vitamin D3, which mediates the action of this vitamin on cells. Upon vitamin D3 binding, VDRB enters the nucleus where it forms heterodimers with the retinoid X receptor (RXR). These VDRB-RXR heterodimers bind to specific response elements on DNA and activate the transcription of vitamin D3-responsive target genes . VDRB is recruited to promoters via its interaction with BAZ1B/WSTF, which mediates the interaction with acetylated histones, an essential step for VDRB-promoter association. It plays a central role in calcium homeostasis and has been shown to impact sensory hair cell development in zebrafish models .

Methodology for studying VDRB function typically involves:

• Gene knockout/knockdown experiments in model organisms

• ChIP-seq analysis to identify DNA binding sites

• Co-immunoprecipitation studies to identify protein interaction partners

• Transcriptome analysis following VDRB activation or inhibition

How do VDRB antibodies differ from VDRA antibodies in experimental applications?

VDRA and VDRB represent two distinct vitamin D receptor isoforms with different functions and expression patterns. Research demonstrates that:

Methodologically, researchers should validate antibody specificity via immunoblotting against recombinant VDRA and VDRB proteins to confirm minimal cross-reactivity before experimental application. As demonstrated in research, "The polyclonal anti-Vdra or anti-Vdrb antibody can detect a strong single endogenous Vdra or Vdrb protein band, whose molecular weight is consistent with that of the exogenous Vdra-FLAG or Vdrb-FLAG" .

What are the recommended protocols for VDRB antibody validation?

Thorough validation is critical for ensuring reliable experimental results with VDRB antibodies. A comprehensive validation approach should include:

• Western blot analysis: Compare antibody reactivity against recombinant VDRB protein, wild-type tissue/cells, and VDRB knockout/knockdown samples.

• Immunoprecipitation specificity: Perform IP followed by mass spectrometry to confirm the identity of precipitated proteins.

• Cross-reactivity assessment: Test against related protein family members, particularly VDRA, to determine specificity.

• Functional validation: Verify that antibody-based detection correlates with functional assays of VDRB activity.

• Epitope mapping: Determine the specific region of VDRB recognized by the antibody to predict potential cross-reactivity.

As demonstrated in research, antibody specificity verification should begin with immunoblotting to confirm the antibody detects "a strong single endogenous VDRB protein band" with the expected molecular weight (approximately 47,663 Da for zebrafish VDRB) .

How should researchers design experiments to study VDRB-RXR interactions using antibody-based approaches?

Studying VDRB-RXR heterodimer formation and function requires carefully designed antibody-based experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-VDRB antibodies to precipitate protein complexes from cells/tissues treated with vitamin D3

  • Detect RXR in immunoprecipitates using anti-RXR antibodies

  • Include appropriate controls: IgG control, VDRB-knockout samples, and vitamin D3-free conditions

• Proximity Ligation Assay (PLA):

  • Employ antibodies against both VDRB and RXR

  • This technique visualizes protein-protein interactions in situ with high sensitivity

  • Quantify interaction signals across different experimental conditions

• Chromatin Immunoprecipitation (ChIP):

  • Use sequential ChIP (ChIP-reChIP) with anti-VDRB followed by anti-RXR antibodies

  • This confirms co-localization of both proteins at specific DNA sites

  • Analyze binding sites through sequencing or qPCR of known vitamin D response elements

Research has established that "Vdra interacted with Rxrbb in the presence of VitD" and this interaction can be detected through co-IP assays, demonstrating the feasibility of antibody-based approaches for studying these interactions .

What are the optimal conditions for using VDRB antibodies in immunohistochemistry applications?

Successful immunohistochemical (IHC) detection of VDRB requires optimization of several key parameters:

• Fixation protocol optimization:

  • Test multiple fixatives (4% PFA, methanol, acetone)

  • Fixation time significantly impacts epitope accessibility

  • For VDRB detection in zebrafish tissues, 4% PFA for 24 hours at 4°C has shown good results

• Antigen retrieval methods comparison:

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

  • Enzymatic retrieval using proteinase K

  • Comparison studies suggest HIER typically yields better results for nuclear receptors like VDRB

• Antibody dilution and incubation optimization:

  • Perform titration experiments (typically 1:100 to 1:1000 range)

  • Test both overnight incubation at 4°C and shorter incubations at room temperature

  • Include known positive and negative control tissues

• Detection system selection:

  • For low abundance targets like VDRB, tyramide signal amplification may improve sensitivity

  • Compare DAB vs. fluorescent detection methods

• Blocking optimization:

  • Test various blocking solutions (BSA, normal serum, commercial blockers)

  • Include steps to block endogenous peroxidase and biotin if applicable

Research examining VDRB expression in zebrafish tissues has demonstrated successful detection when using properly validated antibodies with optimized protocols .

How can researchers effectively combine VDRB antibodies with other techniques for comprehensive pathway analysis?

Integrating VDRB antibody-based detection with complementary techniques provides more robust insights into vitamin D signaling:

• Antibody-ChIP-seq integration:

  • Use ChIP-seq with VDRB antibodies to identify genome-wide binding sites

  • Combine with RNA-seq to correlate binding with transcriptional changes

  • Use motif analysis to identify co-regulatory factors

  • This approach has revealed that "VDR-RXR heterodimers bind to specific response elements on DNA and activate the transcription of vitamin D3-responsive target genes"

• Proximity-dependent labeling:

  • Engineer BioID or APEX2 fusions with VDRB

  • Use antibodies to validate interactions identified by mass spectrometry

  • This reveals the proximal protein network of VDRB in living cells

• Single-cell analysis:

  • Combine VDRB antibodies with single-cell technologies

  • Correlate VDRB expression/localization with cellular phenotypes

  • Analyze cell-specific responses to vitamin D treatment

• Spatial transcriptomics integration:

  • Use VDRB antibodies for tissue localization

  • Correlate with spatial transcriptomic data to understand tissue-specific responses

  • Create comprehensive maps of vitamin D response elements

• Live-cell imaging:

  • Use fluorescently labeled antibody fragments to track VDRB dynamics

  • Correlate with fluorescent reporter assays for vitamin D response elements

Research utilizing these integrated approaches has demonstrated that VDRB forms functional heterodimers that can significantly impact gene expression patterns in response to vitamin D stimulation .

How does VDRB interact with viral infection pathways, and what methodological approaches best capture this relationship?

Recent research has uncovered intriguing connections between VDRB and viral infection responses, particularly in fish models:

These findings suggest important roles for VDRB in modulating antiviral responses, potentially through interaction with viral proteins and regulation of immune signaling pathways.

What are the key considerations when investigating VDRB and VDRA heterodimer formation using antibody-based approaches?

Studying VDRB-VDRA heterodimer formation presents unique challenges requiring specialized antibody-based methodologies:

• Optimization of co-immunoprecipitation protocols:

  • Use of mild detergents to preserve protein-protein interactions

  • Cross-linking optimization to capture transient interactions

  • Sequential immunoprecipitation approaches using antibodies against both proteins

  • Research has shown that "in the absence of VitD, grass carp Vdrb formed a weak heterodimer with Vdra," but "in the presence of VitD, the formation of the Vdra-Vdrb complex was obviously increased"

• Specificity validation strategies:

  • Comparison of immunoprecipitation results using different epitope-targeting antibodies

  • Use of tagged recombinant proteins to verify antibody specificity

  • Implementation of knockout/knockdown controls to confirm specificity

  • Researchers verified antibody specificity using approaches where "the polyclonal anti-Vdra or anti-Vdrb antibody can detect a strong single endogenous Vdra or Vdrb protein band"

• Quantitative assessment approaches:

  • Titration of vitamin D concentration to determine dose-response relationships

  • Time-course experiments to capture kinetics of heterodimer formation

  • Quantitative immunoblotting with appropriate controls and standards

  • Research demonstrated that "the double knockdown of grass carp vdra and vdrb in the presence of VitD showed the strongest protective effect against GCRV infection with the lowest viral load"

• Functional validation methods:

  • Reporter assays to assess heterodimer transcriptional activity

  • ChIP-seq to identify differential binding patterns of heterodimers vs. homodimers

  • Mutagenesis studies to identify critical residues for heterodimer formation

These methodological considerations are essential for accurately characterizing the complex interplay between VDRA and VDRB in vitamin D signaling and their potential roles in disease contexts.

How can researchers interpret contradictory data regarding VDRB function in different model systems?

Resolving contradictory findings about VDRB function across different model systems requires systematic analysis:

• Critical factors in reconciling contradictory data:

  • Species-specific differences in VDRB structure and function

  • Differential expression patterns across tissues and developmental stages

  • Technical variations in antibody specificity and experimental approaches

  • Context-dependent interactions with cofactors

• Systematic approach to resolving contradictions:

  • Cross-validation using multiple antibodies targeting different epitopes

  • Comparison of knockout/knockdown phenotypes across model systems

  • Analysis of evolutionary conservation of VDRB function

  • Investigation of isoform-specific functions and expression patterns

• Case study: Contradictory roles in viral infection:

  • Research shows that "VitD inhibited GCRV replication, but grass carp Vdra or Vdrs promoted GCRV replication"

  • This apparent contradiction was resolved through dose-dependent studies showing that "the negative regulation of grass carp Vdra or Vdrs during GCRV infection is completely abolished by transfection with 2 to ~200 ng VitD"

  • Further research revealed that "VitD also activates a Vdra-Vdrb heterodimer as a corepressor, which attenuates the transcriptions of the RLR antiviral signaling pathway induced by VitD"

• Data integration approaches:

  • Meta-analysis of published literature with attention to methodological differences

  • Structured comparison of experimental conditions across studies

  • Development of unified models that account for context-dependent functions

Researchers should exercise caution when extrapolating findings between species, as functions may be species-specific. For example, while "mammalian VDR interacted directly with the VDRE cluster in the core promoter of hepatitis B virus (HBV) to suppress virus activity," in fish models, VDRB may have different effects on viral replication .

What strategies can overcome antibody cross-reactivity issues between VDRA and VDRB?

Cross-reactivity between VDRA and VDRB antibodies represents a significant technical challenge that requires specific strategies to ensure experimental validity:

• Epitope selection and antibody design:

  • Target non-conserved regions between VDRA and VDRB

  • Use bioinformatic analysis to identify unique epitope sequences

  • Consider generating antibodies against synthetic peptides representing unique regions

  • Perform extensive sequence alignments to predict potential cross-reactivity

• Validation strategies to confirm specificity:

  • Parallel testing against recombinant VDRA and VDRB proteins

  • Use of knockout/knockdown validation in appropriate cell lines

  • Competition assays with purified proteins to determine specificity

  • Immunoblotting against tissue samples from knockout models

• Technical approaches for addressing cross-reactivity:

  • Pre-absorption of antibodies with the cross-reactive protein

  • Optimization of antibody concentration to minimize cross-reactivity

  • Use of highly specific detection methods (e.g., monoclonal antibodies)

  • Implementation of careful controls in each experiment

• Data interpretation considering cross-reactivity:

  • Quantitative assessment of cross-reactivity percentages

  • Statistical approaches to account for known cross-reactivity

  • Multiple antibody use to confirm findings

  • Integration of complementary non-antibody-based approaches

Effective validation approaches should confirm that antibodies can "detect a strong single endogenous Vdra or Vdrb protein band, whose molecular weight is consistent with that of the exogenous Vdra-FLAG or Vdrb-FLAG" as demonstrated in published research.

How should researchers design experiments to investigate VDRB antibody-dependent enhancement in viral infection models?

Investigating potential antibody-dependent enhancement (ADE) involving VDRB requires careful experimental design:

• Experimental design considerations:

  • Use of appropriate viral models known to exhibit ADE phenomena

  • Inclusion of relevant cell types expressing FcγR receptors

  • Careful titration of antibody concentrations to identify enhancement zones

  • Implementation of appropriate positive controls (known ADE-inducing antibodies)

• Key methodological approaches:

  • In vitro viral infection assays with and without VDRB antibodies

  • Flow cytometry to quantify infection rates in target cells

  • Confocal microscopy to visualize internalization of virus-antibody complexes

  • Quantitative RT-PCR to measure viral replication

• Critical controls required:

  • Fc receptor blocking experiments to confirm mechanism

  • Fab fragment controls to assess Fc-independent effects

  • Isotype-matched control antibodies

  • FcγR-negative cell controls

• Interpretation challenges:

  • Distinguishing enhancement from natural infection progression

  • Accounting for antibody neutralization effects

  • Evaluating concentration-dependent effects

Understanding the relationship between VDRB, vitamin D signaling, and viral infection is complex, as research shows that "VitD enhances innate antiviral immunity, whereas overexpression of grass carp vdra or vdrb impairs innate antiviral immunity" . This suggests that antibody-based manipulation of VDRB might have unexpected effects on viral pathogenesis.

Research on antibody-dependent enhancement in other contexts has shown that "antibody-dependent enhancement (ADE) of disease is a general concern for the development of vaccines and antibody therapies because the mechanisms that underlie antibody protection against any virus have a theoretical potential to amplify the infection or trigger harmful immunopathology" .

What are the optimal approaches for developing highly specific monoclonal antibodies against VDRB for advanced research applications?

Developing highly specific monoclonal antibodies against VDRB requires sophisticated approaches:

• Antigen design strategies:

  • Structure-based epitope selection targeting unique VDRB regions

  • Use of multiple peptide antigens representing different domains

  • Production of recombinant VDRB fragments with proper folding

  • Incorporation of post-translational modifications if relevant

• Advanced screening methodologies:

  • Multi-parameter screening against both VDRA and VDRB

  • Application of LIBRA-seq technology which "enables researchers to map the unique sequence of amino acids that make up the reactive portion of an antibody and match it to the specificity"

  • High-throughput SPR or BLI to determine binding kinetics

  • Functional screening assays to identify antibodies that detect physiologically relevant conformations

• Clonal selection optimization:

  • Deep sequencing of hybridoma populations

  • Selection based on both affinity and specificity metrics

  • Epitope binning to identify antibodies against distinct regions

  • Humanization considerations for potential therapeutic applications

• Validation framework for specificity:

  • CRISPR knockout validation in relevant cell systems

  • Cross-species reactivity testing to assess evolutionary conservation

  • Assessment across multiple applications (WB, IP, IHC, etc.)

  • Comparison with existing commercial and research antibodies

Recent advances in antibody technology have yielded methods to identify "rare antibodies that can target a wide range of different viruses" with "exceptional breadth of pathogen coverage" , suggesting that similar approaches could be applied to developing highly specific VDRB antibodies with minimal cross-reactivity to related proteins.

How might new antibody engineering technologies enhance VDRB research in neurodevelopmental disorders?

Emerging antibody technologies offer promising avenues for investigating VDRB's role in neurodevelopment:

• Application of novel antibody formats:

  • Bispecific antibodies targeting VDRB and neural markers simultaneously

  • Brain-penetrant antibody fragments for in vivo studies

  • Intrabodies for tracking VDRB in living neurons

  • Nanobodies for super-resolution microscopy of VDRB localization

• Research opportunities in neurodevelopment:

  • Investigation of VDRB's role in neural patterning and differentiation

  • Assessment of vitamin D signaling in neuronal migration

  • Studies of VDRB in synaptic plasticity and circuit formation

  • Research has shown that "zebrafish embryos lacking vdrb produced fewer sensory hair cells in the ears and showed disruption of balance and motor coordination"

• Technical advances enabling new research:

  • Spatially resolved antibody-based proteomics in brain tissue

  • Antibody-based manipulation of VDRB function in specific neural populations

  • CRISPR-engineered models with antibody-taggable VDRB

  • Application of Design of Experiments (DOE) approaches to optimize antibody parameters for neuroscience applications

• Translational research potential:

  • Correlation of VDRB dysfunction with neurodevelopmental disorders

  • Development of antibody-based diagnostics for vitamin D signaling abnormalities

  • Therapeutic antibody engineering targeting VDRB-mediated pathways

These approaches could significantly advance understanding of VDRB's neural functions, particularly given the growing evidence for vitamin D's role in brain development and function.

What are the emerging methodologies for studying VDRB antibody conformational dynamics and their impact on receptor function?

Advanced biophysical and computational approaches are revolutionizing our understanding of antibody-receptor interactions:

• Cutting-edge structural biology approaches:

  • Cryo-electron microscopy of VDRB-antibody complexes

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • Single-molecule FRET to monitor conformational changes upon antibody binding

  • NMR studies of antibody effects on VDRB dynamics

• Computational modeling advances:

  • Molecular dynamics simulations of antibody-VDRB interactions

  • In silico epitope mapping and binding prediction

  • Machine learning approaches for antibody design

  • Recent research has demonstrated the value of "machine learning, computational biology, drug development, and software engineering to transform the pace, cost, and scale of antibody discovery"

• Functional assessment methodologies:

  • Surface plasmon resonance to quantify binding kinetics

  • Thermal shift assays to assess stabilization/destabilization effects

  • HTRF/TR-FRET assays to monitor coregulator recruitment

  • Cell-based assays with conformation-specific reporters

• Integration with gene editing approaches:

  • CRISPR-mediated introduction of conformational biosensors

  • Structure-guided mutagenesis to test computational predictions

  • Creation of synthetic VDRB variants with altered antibody binding properties

Understanding how antibodies affect VDRB conformation could lead to the development of conformation-selective antibodies that specifically modulate distinct VDRB functions, potentially offering new therapeutic approaches for vitamin D-related disorders.

How can VDRB antibody research contribute to understanding the evolutionary conservation of vitamin D signaling across species?

Comparative VDRB antibody studies across species can provide valuable evolutionary insights:

• Cross-species reactivity assessment approaches:

  • Systematic testing of VDRB antibodies across evolutionary diverse species

  • Epitope conservation analysis using sequence alignment tools

  • Development of pan-species antibodies targeting highly conserved regions

  • Species-specific antibodies for divergent regions

• Evolutionary questions addressable through antibody research:

  • When did distinct VDRA and VDRB isoforms emerge?

  • How conserved are VDRB interactions with RXR across species?

  • Are there species-specific VDRB cofactors identifiable through immunoprecipitation?

  • Research has shown significant differences between mammalian and fish VDR responses, as "mammalian VDR interacted directly with the VDRE cluster in the core promoter of hepatitis B virus (HBV) to suppress virus activity" while fish VDRB may have different effects

• Methodological approaches for evolutionary studies:

  • Immunohistochemical mapping of VDRB expression across diverse species

  • Comparative ChIP-seq to identify conserved and divergent binding sites

  • Antibody-based affinity purification followed by mass spectrometry to identify species-specific interaction partners

  • Functional validation of conserved epitopes through cross-species antibody testing

• Potential insights from evolutionary antibody research:

  • Identification of core conserved functions versus species-specific adaptations

  • Understanding of vitamin D signaling evolution in relation to habitat and diet

  • Correlation of VDRB structural changes with functional adaptations

These studies could significantly enhance our understanding of how vitamin D signaling has evolved and adapted across diverse species, potentially revealing fundamental insights into conserved mechanisms of nuclear receptor function.