Phospho-VDR (Ser51) Antibody

What is Phospho-VDR (Ser51) Antibody and what does it specifically detect?

Phospho-VDR (Ser51) antibody is a specialized immunological reagent that recognizes the Vitamin D Receptor (VDR) protein exclusively when phosphorylated at serine residue 51. VDR functions as a nuclear hormone receptor for vitamin D3 and belongs to the family of trans-acting transcriptional regulatory factors with sequence similarity to steroid and thyroid hormone receptors . This antibody specifically detects endogenous levels of VDR protein only when phosphorylated at the Ser51 position, making it valuable for studying this particular post-translational modification . The antibodies are typically generated by immunizing rabbits with synthesized peptides derived from human VDR around the phosphorylation site of Ser51, usually spanning amino acids 16-65 .

What applications is Phospho-VDR (Ser51) antibody suitable for?

According to manufacturer specifications, Phospho-VDR (Ser51) antibodies are validated for multiple research applications:

These applications enable researchers to detect and quantify phosphorylated VDR across various experimental platforms and sample types .

What are the optimal storage and handling conditions for Phospho-VDR (Ser51) antibodies?

For maximum antibody stability and performance, Phospho-VDR (Ser51) antibodies should be stored at -20°C . It is critical to avoid repeated freeze-thaw cycles as these can significantly reduce antibody efficacy and specificity . Most commercially available Phospho-VDR (Ser51) antibodies are formulated in PBS buffer containing 50% glycerol, 0.02% sodium azide, and sometimes 0.5% BSA or protective protein at pH 7.4 . This formulation helps maintain stability during storage while preventing microbial contamination.

What is the biological significance of VDR phosphorylation at Ser51?

VDR phosphorylation at Ser51 plays a complex role in regulating vitamin D signaling. This serine residue is located between the two zinc fingers of VDR and serves as a substrate for protein kinase C-β (PKC-β) . The functional consequences of this phosphorylation have been investigated through various mutation studies:

• Mutation of Ser51 to glycine (S51G) abolished PKC-β-catalyzed phosphorylation and led to a 65% decrease in transcriptional activity compared to wild-type VDR

• Replacement with aspartic acid (S51D, mimicking negative charge) resulted in a 90% decrease in transcriptional activity

• Substitution with threonine (S51T) led to a 55% decrease in activity

Interestingly, research has revealed contradictory findings regarding the precise role of this phosphorylation. While some data suggest a positive role in 1,25(OH)₂D-dependent transcriptional activation, other experiments indicate that incubation of E. coli-expressed VDR with PKC-β decreased its ability to bind to the vitamin D response element (VDRE), suggesting a potentially inhibitory effect . This apparent contradiction highlights the complexity of VDR regulation and suggests that the effect of Ser51 phosphorylation may be context-dependent.

How does PKC-β specifically interact with and phosphorylate VDR at Ser51?

Protein Kinase C-β (PKC-β) has been identified as the specific kinase responsible for phosphorylating VDR at the Ser51 position . The amino acid sequence context surrounding Ser51 (51MKRK located between the two zinc fingers) creates a recognition motif for PKC-β . Through detailed mutation studies, researchers have confirmed that Ser51 is the sole target of PKC-β-mediated phosphorylation in human VDR, as other potential serine residues (Ser119 and Ser125) were not confirmed as phosphorylation sites for this kinase .

In experimental settings, treatment of cells with phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, enhances phosphorylation of wild-type VDR but has no effect on the Ser51 mutant VDR . This further confirms the specificity of this phosphorylation site in cellular contexts. The PKC pathway thus provides a mechanism linking cellular signaling events related to growth regulation and tumor promotion to the phosphorylation and function of VDR .

How can researchers validate the specificity of Phospho-VDR (Ser51) antibodies?

Robust validation of phospho-specific antibodies is essential for reliable research outcomes. Several complementary approaches can confirm the specificity of Phospho-VDR (Ser51) antibodies:

• Peptide competition assays: Preincubating the antibody with the synthesized phosphopeptide immunogen should block specific binding. Search result demonstrates this approach, showing that treatment with the synthesized peptide blocks antibody recognition in Western blot.

• Mutation studies: Comparing antibody recognition between wild-type VDR and a S51A mutant (where serine is replaced with alanine, preventing phosphorylation) should show detection of only the wild-type protein.

• Phosphatase treatment: Treating samples with lambda phosphatase to remove phosphate groups should eliminate or significantly reduce signal if the antibody is truly phospho-specific.

• Stimulation experiments: Treating cells with PKC activators should increase signal intensity, while PKC inhibitors should decrease it, if the antibody specifically recognizes the PKC-mediated phosphorylation site.

• Correlation with other techniques: Orthogonal methods such as mass spectrometry can provide additional confirmation of phosphorylation at the Ser51 position.

What are optimal experimental conditions for detecting phosphorylated VDR at Ser51?

Based on published research methodologies, the following conditions optimize detection of phosphorylated VDR at Ser51:

For Western blotting:

• Cell models: COLO cells appear suitable for detecting phospho-VDR

• Stimulation: Treatment with insulin (0.01U/ml for 15 minutes) has been shown to enhance phosphorylation in COLO cells

• Sample preparation: Include phosphatase inhibitors in lysis buffers to prevent dephosphorylation during extraction

• Electrophoresis parameters: Phospho-VDR typically appears at approximately 38 kDa (though calculated MW is noted as 48 kDa in some sources)

• Antibody dilution: 1:500-1:2000 is typically optimal

For IHC and IF:

• Tissue preparation: Paraffin-embedded sections with appropriate antigen retrieval

• Antibody dilution: 1:100-1:300 for IHC and 1:50-1:200 for IF

• Controls: Include positive controls (tissues known to express phosphorylated VDR) and negative controls (phosphatase-treated sections)

How do mutations at the Ser51 site affect VDR function and antibody recognition?

Mutations at the Ser51 site have revealed important insights about both VDR function and considerations for antibody-based detection:

Effects on VDR function:

• S51G mutation: 65% decrease in transcriptional activity compared to wild-type VDR

• S51D mutation (phosphomimetic): 90% decrease in transcriptional activity

• S51T mutation: 55% decrease in activity

• S51A mutation: Eliminated PKC-β phosphorylation but surprisingly retained wild-type VDR transactivation capacity and DNA binding ability

These findings suggest that both the presence of serine at position 51 and its potential for phosphorylation play complex roles in VDR function that cannot be simply categorized as activating or inhibitory.

Effects on antibody recognition:

• Phospho-specific antibodies should not recognize S51A or S51G mutants

• According to manufacturer specifications, these antibodies detect VDR "only when phosphorylated at S51"

• This specificity makes these antibodies valuable tools for studying the dynamics of this post-translational modification

How does Ser51 phosphorylation compare to other phosphorylation sites in VDR and related proteins?

While the search results focus primarily on Ser51 phosphorylation in VDR, this site can be compared to phosphorylation sites in other proteins:

What are the current contradictions or knowledge gaps regarding VDR Ser51 phosphorylation?

Several contradictions and knowledge gaps exist in current research on VDR Ser51 phosphorylation:

• Contradictory functional effects: Some studies suggest phosphorylation at Ser51 positively regulates VDR activity, while others indicate a negative influence on transcriptional function . The observation that S51A mutation eliminates phosphorylation but retains wild-type activity further complicates this picture.

• Context dependency: The effect of Ser51 phosphorylation may vary depending on:

  • Cell type and tissue context

  • Presence of cofactors or other post-translational modifications

  • Specific transcriptional targets being assessed

  • Experimental conditions and methodologies

• Regulatory mechanisms: The upstream signaling pathways that regulate PKC-β-mediated phosphorylation of VDR in different physiological and pathological contexts remain incompletely understood.

• Cross-talk with other modifications: How Ser51 phosphorylation interacts with other post-translational modifications of VDR has not been thoroughly investigated.

• Physiological significance: While biochemical effects have been studied in detail, the physiological importance of this phosphorylation in vitamin D signaling across different tissues remains to be fully elucidated.

What are common technical challenges when working with Phospho-VDR (Ser51) antibodies?

Researchers may encounter several challenges when working with phospho-specific antibodies:

• Variable phosphorylation levels: The proportion of VDR phosphorylated at Ser51 may be low under basal conditions, making detection challenging.

• Phosphatase activity during sample preparation: Phosphate groups can be rapidly lost during sample preparation if appropriate phosphatase inhibitors are not included.

• Antibody specificity issues: Despite claims of specificity, antibodies may exhibit some cross-reactivity with similar phosphorylation motifs in other proteins.

• Variable VDR expression: VDR expression varies considerably between cell types and tissues, affecting detection sensitivity.

• Signal-to-noise ratio: Background signal can be problematic, particularly in immunohistochemistry applications.

What controls should be included when conducting experiments with Phospho-VDR (Ser51) antibodies?

To ensure reliable and interpretable results, the following controls should be incorporated:

Positive controls:

• Cells treated with PKC activators such as phorbol 12-myristate 13-acetate (PMA) or insulin

• In vitro phosphorylated recombinant VDR (using purified PKC-β)

• COLO cells, which have been documented to exhibit detectable Ser51 phosphorylation

Negative controls:

• Samples treated with lambda phosphatase to remove phosphate groups

• Cells treated with PKC inhibitors to reduce phosphorylation

• Expression of S51A mutant VDR that cannot be phosphorylated at position 51

• Peptide competition controls, where the antibody is pre-incubated with the phosphopeptide immunogen