Phospho-RARA (Ser77) Antibody

What is the biological significance of RARA phosphorylation at Serine 77?

Phosphorylation of Retinoic Acid Receptor alpha (RARA) at Serine 77 plays a crucial role in its transcriptional activity. Unlike other phosphorylation sites, Ser77 phosphorylation is specifically critical for the receptor's function in regulating gene expression during various biological processes . Research indicates that while phosphorylation occurs at multiple serine and threonine residues in RARA, the phosphorylation at Ser77 remains relatively constant during the cell cycle but is fundamental for activating transcription of target genes . This post-translational modification affects RARA's ability to recruit coactivators and initiate transcription after ligand binding, making it an essential regulatory mechanism for retinoic acid signaling pathways .

The Phospho-RARA (Ser77) antibody demonstrates cross-reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species . This reactivity is attributed to the conservation of the amino acid sequence surrounding the Ser77 phosphorylation site within these species. When designing experiments, researchers should consider that although the antibody has been validated in these three species, sensitivity may vary depending on the experimental conditions and sample preparation methods. For work with other species, preliminary validation studies are recommended to confirm cross-reactivity before proceeding with full-scale experiments .

What are the recommended storage conditions for maintaining antibody activity?

For optimal performance and stability, Phospho-RARA (Ser77) antibody should be:

• Shipped at 4°C but stored at -20°C upon receipt

• Aliquoted to avoid repeated freeze-thaw cycles, which significantly reduce antibody activity

• Stored in the provided formulation containing 50% glycerol, which prevents freezing at -20°C and maintains antibody stability

• Maintained in a buffer containing sodium azide (0.02%) to prevent microbial contamination

• Expected to remain stable for approximately 12 months from the date of receipt when properly stored

Researchers should avoid exposing the antibody to elevated temperatures or prolonged periods at room temperature, as these conditions can accelerate degradation and reduce specificity .

How can researchers optimize Western blot protocols for Phospho-RARA (Ser77) detection?

To achieve optimal results when using Phospho-RARA (Ser77) antibody in Western blot applications:

• Sample preparation: Use phosphatase inhibitors during protein extraction to preserve phosphorylation status

• Loading control: Include both phosphorylated and non-phosphorylated controls to verify specificity

• Blocking: Use 5% BSA rather than milk, as milk contains phosphoproteins that may interfere with phospho-specific antibody binding

• Primary antibody incubation: Start with a 1:1000 dilution in 5% BSA/TBST and incubate overnight at 4°C

• Expected band size: Look for a band at approximately 50 kDa (reported molecular weight of RARA)

• Validation: Consider using lambda phosphatase-treated samples as negative controls to confirm phospho-specificity

For challenging samples with low phospho-RARA expression, signal enhancement systems or longer exposure times may be necessary .

How does RARA phosphorylation at Ser77 integrate with other post-translational modifications?

RARA undergoes multiple post-translational modifications that create a complex regulatory network. Phosphorylation at Ser77 operates within this network as follows:

• Phosphorylation profile: Besides Ser77, RARA is phosphorylated at Ser219 and Ser369 by PKA, which affects ligand binding and nuclear localization

• Relationship with sumoylation: RARA is primarily sumoylated with SUMO2 at Lys-399, but all-trans retinoic acid (ATRA) binding induces a conformational change allowing additional sumoylation at Lys-166 and Lys-171

• Interaction with ubiquitination: RARA is ubiquitinated by UBR5, leading to degradation when not associated with coactivators (NCOAs)

• Influence on acetylation: Acetylation of RARA increases upon pulsatile shear stress and decreases with oscillatory shear stress

When investigating the functional significance of Ser77 phosphorylation, researchers should consider these interrelated modifications that collectively determine RARA activity, localization, and stability .

What experimental approaches can elucidate the kinase responsible for RARA Ser77 phosphorylation?

To identify and validate the kinase(s) responsible for RARA Ser77 phosphorylation:

• In silico prediction: Use phosphorylation site prediction tools to identify candidate kinases based on the amino acid sequence surrounding Ser77

• Kinase inhibitor screen: Treat cells with specific kinase inhibitors and assess changes in Ser77 phosphorylation using the Phospho-RARA (Ser77) antibody

• In vitro kinase assays: Perform assays with recombinant RARA and purified candidate kinases

• Genetic approaches: Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout of candidate kinases

• Phospho-mapping: Conduct mass spectrometry analysis after in vitro phosphorylation reactions

How does RARA phosphorylation at Ser77 influence its function in different cellular contexts?

RARA phosphorylation at Ser77 has context-dependent functions that vary across cell types and physiological processes:

• Spermatogenesis: RARA plays an essential role in retinoic acid-induced germ cell development, with phosphorylation potentially regulating the survival of early spermatocytes at the beginning of meiosis

• Skeletal development: In concert with RARG, RARA is required for skeletal growth, matrix homeostasis, and growth plate function, with phosphorylation potentially modulating these activities

• Vascular endothelium: Together with RXRA, RARA regulates microRNA-10a expression, influencing the GATA6/VCAM1 signaling response to pulsatile shear stress

• Inflammatory response: In association with HDAC3, HDAC5, and HDAC7 corepressors, RARA represses microRNA-10a and promotes inflammatory responses

• Granulopoiesis: RARA mediates retinoic acid-induced granulopoiesis, with phosphorylation potentially modulating this developmental process

When designing experiments to study phosphorylation-dependent functions, researchers should select cellular models relevant to these specific contexts and incorporate appropriate functional readouts .

What controls should be included when validating Phospho-RARA (Ser77) antibody specificity?

To rigorously validate the specificity of Phospho-RARA (Ser77) antibody:

• Phosphatase treatment: Treat half of your sample with lambda phosphatase to remove phosphate groups

• Competing peptide: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides corresponding to the Ser77 region

• RARA knockdown/knockout: Use siRNA or CRISPR to reduce or eliminate RARA expression

• Stimulation/inhibition: Treat cells with stimuli known to increase or decrease RARA phosphorylation

• Isotype control: Include a matched isotype control (rabbit IgG) at equivalent concentration

• Total RARA detection: Run parallel experiments with an antibody detecting total RARA regardless of phosphorylation status

These controls help distinguish between specific signals and background, ensuring reliable interpretation of experimental results .

How can researchers troubleshoot weak or non-specific signals when using Phospho-RARA (Ser77) antibody?

When encountering signal problems with Phospho-RARA (Ser77) antibody:

For weak signals:

• Optimize protein extraction with phosphatase inhibitors to preserve phosphorylation

• Increase antibody concentration (within recommended range)

• Extend incubation time or adjust temperature

• Enhance detection using more sensitive substrates

• Enrich for nuclear proteins, as RARA primarily localizes to the nucleus

For non-specific signals:

• Optimize blocking conditions (5% BSA recommended for phospho-specific antibodies)

• Increase washing duration and frequency

• Reduce antibody concentration

• Pre-adsorb antibody with non-specific proteins

• Confirm sample integrity and eliminate proteolytic degradation

If performing immunohistochemistry, optimize antigen retrieval methods as phospho-epitopes may be particularly sensitive to fixation conditions .

What methodological considerations are important when studying RARA signaling dynamics?

To effectively investigate RARA signaling dynamics using Phospho-RARA (Ser77) antibody:

• Temporal resolution: Design time-course experiments to capture rapid changes in phosphorylation status

• Subcellular localization: Combine with fractionation or immunofluorescence to track phospho-RARA localization

• Protein-protein interactions: Use co-immunoprecipitation with Phospho-RARA (Ser77) antibody to identify phosphorylation-dependent interacting partners

• Chromatin association: Implement ChIP assays to determine how Ser77 phosphorylation affects RARA binding to retinoic acid response elements (RAREs)

• Transcriptional output: Correlate phosphorylation status with expression of RARA target genes

• Signal integration: Investigate cross-talk between retinoic acid signaling and other pathways that might influence RARA phosphorylation

Remember that RXR/RAR heterodimers bind to RAREs composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5, and this binding is regulated by conformational changes influenced by phosphorylation status .

How might Phospho-RARA (Ser77) antibody contribute to leukemia research?

Phospho-RARA (Ser77) antibody offers significant potential for investigating acute promyelocytic leukemia (APL), as aberrations involving RARA are implicated in this disease :

• Diagnostic applications: Distinguish between phosphorylation patterns in normal versus leukemic cells

• Therapeutic monitoring: Track changes in RARA phosphorylation during treatment with retinoids or other therapies

• Mechanistic studies: Investigate how fusion proteins (like PML-RARA) affect phosphorylation status and subsequent cellular functions

• Biomarker development: Evaluate whether phosphorylation at Ser77 correlates with disease progression or treatment response

• Drug discovery: Screen compounds that specifically modulate RARA phosphorylation as potential therapeutic agents

Researchers should consider using cell lines derived from APL patients alongside primary patient samples to comprehensively characterize the role of Ser77 phosphorylation in leukemogenesis and treatment response .

What techniques can be combined with Phospho-RARA (Ser77) antibody for single-cell analysis?

To achieve single-cell resolution in studying RARA phosphorylation dynamics:

• Single-cell immunofluorescence: Optimize staining protocols using Phospho-RARA (Ser77) antibody with high-resolution confocal microscopy

• Flow cytometry: Develop intracellular staining protocols for quantitative analysis of phospho-RARA levels across cell populations

• Mass cytometry (CyTOF): Label Phospho-RARA (Ser77) antibody with heavy metals for multiplexed signaling analysis

• Imaging mass cytometry: Combine tissue imaging with single-cell resolution phospho-protein analysis

• Proximity ligation assays: Detect interactions between phosphorylated RARA and other proteins at single-molecule resolution

• Single-cell Western blot: Adapt protocols for detecting phospho-RARA in individual cells

These approaches enable researchers to investigate heterogeneity in RARA phosphorylation within complex tissues or cell populations responding to retinoic acid or other stimuli .