VDR (Ab-51) Antibody

What is VDR (Ab-51) Antibody and what epitope does it recognize?

VDR (Ab-51) Antibody is a polyclonal antibody raised in rabbits against a synthetic non-phosphopeptide derived from Human Vitamin D3 Receptor. The epitope is specifically centered around the phosphorylation site of serine 51 (R-R-S(p)-M-K) of the VDR protein, allowing the antibody to detect endogenous levels of total Vitamin D3 Receptor protein . This recognition site is significant because the region around serine 51 is involved in regulation of VDR activity, making this antibody particularly useful for studying VDR function and regulation in experimental contexts.

The antibody's specificity for the Ser51 region distinguishes it from other VDR antibodies that may target different epitopes, offering researchers a tool to investigate specific aspects of VDR biology. When selecting an antibody for VDR detection, understanding the target epitope is essential for experimental design and interpretation of results.

What are the recommended storage and handling conditions for VDR (Ab-51) Antibody?

For optimal preservation of antibody activity, VDR (Ab-51) Antibody should be stored at -20°C or -80°C upon receipt . Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce sensitivity in experimental applications. The antibody is supplied in a stabilization buffer consisting of rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

When handling the antibody for experiments, aliquoting into single-use volumes is strongly recommended to minimize freeze-thaw cycles. Working dilutions should be prepared fresh before use and can typically be stored at 4°C for up to one week, though this may vary based on experimental conditions and should be validated by each laboratory.

What applications has VDR (Ab-51) Antibody been validated for?

VDR (Ab-51) Antibody has been validated for the following research applications:

For Western blot applications, the antibody has successfully detected VDR in Jurkat cell extracts, demonstrating its utility in analyzing VDR expression in cell culture models . When planning experiments, researchers should note that the optimal dilution may need to be determined empirically for each specific experimental system and application.

It's important to emphasize that this antibody is intended "For Research Use Only" and is not validated for diagnostic or therapeutic procedures .

What is the recommended protocol for using VDR (Ab-51) Antibody in Western Blot analysis?

Based on published methodologies using VDR antibodies, the following protocol is recommended for Western Blot analysis with VDR (Ab-51) Antibody:

• Sample Preparation:

  • Lyse cells or tissue samples in an appropriate lysis buffer containing protease inhibitors

  • Centrifuge lysates and transfer supernatant to fresh tubes

  • Measure protein concentration using a standard assay (e.g., Bradford or BCA)

• SDS-PAGE and Transfer:

  • Load 20-60 μg of protein per lane on a 10% SDS-PAGE gel

  • Separate proteins by electrophoresis

  • Transfer proteins to a polyvinylidene fluoride (PVDF) membrane

• Antibody Incubation:

  • Block membrane with 5% skim milk powder in PBS containing 0.1% Tween 20

  • Incubate with VDR (Ab-51) Antibody at a dilution of 1:500 to 1:3000 (optimize for your specific sample) overnight at 4°C

  • Wash membrane thoroughly with PBS-T

  • Incubate with HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:1000 dilution for 1 hour at room temperature

• Detection:

  • Visualize bands using enhanced chemiluminescence reagent

  • Analyze band intensity using appropriate imaging software such as ImageJ

This protocol is based on successful detection of VDR in Jurkat cells and should be optimized for specific research contexts. Researchers should include appropriate positive and negative controls to validate antibody specificity.

How can I troubleshoot weak or inconsistent signals when using VDR (Ab-51) Antibody?

When encountering weak or inconsistent signals with VDR (Ab-51) Antibody, consider the following methodological adjustments:

For tissues or cells with low endogenous VDR expression, it's important to note that VDR levels can be difficult to detect. Studies have shown that VDR protein can be readily measured in kidney tissue, while detection in immune tissues like spleen is more challenging due to low expression levels in resting immune cells . Additionally, activation of certain cell types (e.g., T cells) can upregulate VDR expression, making the protein detectable 2-3 days post-activation .

How should samples be prepared for optimal VDR detection in different tissue types?

Sample preparation protocols should be tailored based on tissue type and research questions:

For cell lines (e.g., Jurkat cells):

• Rinse cells with cold PBS

• Lyse in buffer containing 20 mM HEPES (pH 7.4), 0.5% Nonidet P-40, 100 mM NaCl, 1 mM EDTA, and 2 mM Na₃VO₄, supplemented with protease inhibitors

• Incubate on ice for 30 minutes with periodic vortexing

• Centrifuge at 14,000g for 10 minutes at 4°C

• Transfer supernatant to fresh tube and measure protein concentration

For muscle or tissue samples:

• Homogenize samples in RLT solution (QIAGEN) or appropriate lysis buffer

• For RNA extraction, follow with proteinase K treatment and RNA isolation using RNeasy Mini kit

• For protein extraction, centrifuge homogenate and collect supernatant containing soluble proteins

• Measure protein concentration before proceeding to Western blot analysis

When working with tissues known to have low VDR expression, consider enrichment techniques such as immunoprecipitation before Western blot analysis to increase detection sensitivity. Additionally, for immunofluorescence applications, fixation and permeabilization with 3% paraformaldehyde and 0.1% Triton X-100 in PBS for 30 minutes has been successfully used for VDR detection in tissue sections .

How does VDR (Ab-51) Antibody compare to other VDR antibodies in terms of specificity and sensitivity?

The specificity and sensitivity of VDR antibodies has been a subject of scientific debate. Studies comparing various VDR antibodies have yielded conflicting results regarding their specificity in different experimental contexts . While VDR (Ab-51) Antibody targets the serine 51 region, other antibodies like the VDR-D6 antibody (sc13133; Santa Cruz Biotechnology) target different epitopes and have been reported to show specificity in certain contexts .

Research by Wang and colleagues evaluated multiple anti-VDR antibodies and found that the D-6 antibody demonstrated specific staining of VDR in mouse kidney and small intestine tissues, while failing to stain brain tissue by immunohistochemistry . This highlights the importance of antibody validation in specific experimental systems.

When designing critical experiments, researchers should consider:

• Using multiple antibodies targeting different VDR epitopes to confirm findings

• Including appropriate positive controls (tissues with known high VDR expression like kidney or duodenum)

• Including negative controls (tissues from VDR knockout models when available)

• Validating antibody specificity through siRNA knockdown or overexpression systems

Can VDR (Ab-51) Antibody detect post-translational modifications of the Vitamin D Receptor?

While VDR (Ab-51) Antibody was raised against a non-phosphopeptide derived from the region surrounding serine 51, its ability to distinguish between phosphorylated and non-phosphorylated forms of VDR requires experimental validation. The antibody is described as detecting "endogenous levels of total Vitamin D3 Receptor protein" , suggesting it recognizes VDR regardless of phosphorylation status at serine 51.

For researchers specifically interested in studying VDR phosphorylation, it's worth noting that:

• Phosphorylation at different residues can affect VDR function and activity

• Specific phospho-antibodies may be required to study these modifications

• Complementary techniques such as mass spectrometry or phosphatase treatments may be necessary to confirm phosphorylation status

Other VDR antibodies specifically targeting other post-translational modifications are available, such as those detecting phosphorylation at serine 208 , which may be more suitable for studying specific modifications of VDR.

What are the latest findings on VDR expression in different tissue types and how can VDR (Ab-51) Antibody contribute to this research?

Recent research has expanded our understanding of VDR expression patterns across different tissues and cell types. Studies using various detection methods, including antibody-based approaches, have provided new insights:

• Skeletal Muscle Expression: There have been conflicting reports about VDR expression in skeletal muscle, with some studies detecting VDR while others did not. This controversy highlights the importance of antibody specificity and sensitive detection methods .

• Immune Cell Expression: Research using VDR reporter mice revealed that immune cells exhibit variable VDR expression patterns. Specifically:

  • Neutrophils and monocytes predominantly express VDR

  • Some immune cells like ILC2 and certain T cell populations may never express VDR

  • T cells can upregulate VDR expression upon activation, with protein detection possible after 72 hours

• Tissue-Specific Expression Levels: VDR is highly and constitutively expressed in the kidney, colon, and small intestine, while expression in immune tissues like the spleen is significantly lower .

VDR (Ab-51) Antibody can contribute to this field by providing researchers with a tool to detect total VDR protein in various experimental systems. Its ability to recognize an epitope around serine 51 offers opportunities to study VDR expression in different contexts, particularly when combined with complementary approaches like qPCR, reporter systems, or other antibodies targeting different epitopes.

How should researchers interpret Western Blot results using VDR (Ab-51) Antibody?

When interpreting Western Blot results with VDR (Ab-51) Antibody, consider the following guidelines:

• Expected Molecular Weight: VDR typically appears as a band at approximately 48-55 kDa. Variations in apparent molecular weight may occur due to post-translational modifications or tissue-specific isoforms.

• Multiple Bands: If multiple bands are observed, consider:

  • Potential degradation products (especially if bands appear below the expected size)

  • Cross-reactivity with related nuclear receptors (validate with knockout controls)

  • VDR isoforms (documented in literature)

  • Post-translational modifications affecting mobility

• Quantification: For accurate quantification of VDR expression:

  • Normalize to appropriate loading controls (e.g., β-actin, GAPDH)

  • Use image analysis software like ImageJ for densitometry

  • Include standard curves with known amounts of purified protein when possible

• Tissue-Dependent Expression: Consider tissue-specific context when interpreting results. VDR expression varies dramatically between tissues, with kidney and intestinal tissues showing high expression, while immune tissues and skeletal muscle may have very low baseline expression that increases upon stimulation .

What experimental designs are recommended for studying VDR signaling pathways using VDR (Ab-51) Antibody?

When designing experiments to investigate VDR signaling pathways, researchers should consider multimodal approaches:

• Stimulus-Response Studies:

  • Treat cells with 1,25-dihydroxyvitamin D3 at physiologically relevant concentrations (typically 1-100 nM)

  • Analyze VDR protein expression, nuclear translocation, and target gene activation in a time-dependent manner

  • Use VDR (Ab-51) Antibody for Western blot analysis of total VDR protein levels

• Protein-Protein Interaction Analysis:

  • Design co-immunoprecipitation experiments using VDR (Ab-51) Antibody to pull down VDR and associated proteins

  • Follow with mass spectrometry to identify novel interaction partners

  • Validate interactions with reverse co-IP and proximity ligation assays

• Chromatin Association Studies:

  • Perform chromatin immunoprecipitation (ChIP) experiments to identify VDR binding sites

  • Note that while VDR (Ab-51) Antibody can be used for Western blot and ELISA, its suitability for ChIP applications would need validation

  • Consider that VDR's interaction with the WINAC complex and BAZ1B/WSTF is essential for VDR-promoter association

• Cell Type-Specific Regulation:

  • When studying immune cells, note that activation can upregulate VDR expression

  • T cell activation leads to detectable VDR protein levels 2-3 days post-activation

  • In macrophages, cytokines or toll-like receptor agonists can upregulate VDR expression

How can researchers integrate VDR (Ab-51) Antibody data with other experimental approaches for comprehensive VDR research?

For comprehensive investigation of VDR biology, integrate antibody-based detection with complementary approaches:

• Transcriptional Analysis:

  • Pair protein detection using VDR (Ab-51) Antibody with mRNA quantification

  • Use real-time quantitative PCR with primers designed for VDR (e.g., forward: 5′-gtggacattggcatgatgaa-3′ and reverse: 5′-ttacgtctgcacgaattgga-3′)

  • Consider RNA-seq for genome-wide expression analysis of VDR target genes

• Functional Validation Techniques:

  • Utilize siRNA or CRISPR-based VDR knockdown/knockout systems

  • Complement with overexpression studies using tagged VDR constructs

  • Consider reporter assays with VDR-responsive elements to measure functional activity

• Genetic Approaches:

  • Leverage VDR knockout models or reporter systems (e.g., VDRCre mice with tdTomato reporters)

  • These systems provide sensitive detection of VDR expression history in cells where protein levels may be below Western blot detection limits

  • Such genetic approaches revealed that peripheral neutrophils and monocytes are almost all VDR-positive, while some immune cells may never express VDR

• Imaging Techniques:

  • For cellular localization studies, use immunofluorescence with VDR antibodies

  • Include DAPI staining to assess nuclear localization

  • Validate specificity with appropriate controls (primary antibody omission, VDR knockout tissues)

By combining these methodological approaches, researchers can overcome the limitations of any single technique and develop a more comprehensive understanding of VDR biology in their experimental system.

What are the considerations for using VDR (Ab-51) Antibody in multi-omics research approaches?

Multi-omics integration is becoming increasingly important in VDR research. When incorporating VDR (Ab-51) Antibody into these approaches, consider:

• Proteomics Integration:

  • VDR (Ab-51) Antibody can be used for immunoprecipitation followed by mass spectrometry to identify VDR interactome

  • The antibody's specificity for the serine 51 region may capture specific subsets of VDR complexes

  • Results should be validated using orthogonal approaches such as proximity ligation assays or FRET analysis

• Epigenomics Connections:

  • VDR functions as a transcription factor that mediates the action of vitamin D3 by controlling hormone-sensitive gene expression

  • Its association with the WINAC chromatin-remodeling complex and interaction with acetylated histones via BAZ1B/WSTF is essential for promoter association

  • Consider ChIP-seq approaches to map genome-wide VDR binding sites in conjunction with protein analysis

• Single-Cell Applications:

  • For single-cell protein analysis, validate antibody performance in flow cytometry or mass cytometry panels

  • Consider the reported detection limits of VDR in different cell types when designing experiments

  • Recent research using genetic reporter systems has revealed cell type-specific VDR expression patterns not previously detectable with antibody-based methods alone