Phospho-EP300 (S89) Antibody

What is EP300 and what role does phosphorylation at S89 play in its function?

EP300 (also known as p300) is an adenovirus E1A-associated cellular transcriptional co-activator protein that functions as a histone acetyltransferase. It is related to CREB-binding protein (CBP) and can stimulate transcription through CREB activation . EP300 also serves as a co-activator of HIF1A (hypoxia-inducible factor 1 alpha), contributing to the regulation of hypoxia-induced genes such as VEGF .

Phosphorylation at serine 89 (S89) represents a post-translational modification that can modulate EP300's activity, protein-protein interactions, and potentially its subcellular localization. This specific phosphorylation site is significant enough to warrant dedicated antibodies for its detection, suggesting its importance in signaling pathways and transcriptional regulation mechanisms .

What are the primary applications for Phospho-EP300 (S89) antibodies in research?

Phospho-EP300 (S89) antibodies are primarily used in several experimental techniques:

• Immunohistochemistry (IHC-P): For detection of phosphorylated EP300 in formalin-fixed paraffin-embedded tissue sections, providing insights into protein expression and localization within tissue context

• Dot Blotting: For rapid detection of the phosphorylated protein without size separation

• ELISA: For quantitative detection of phosphorylated EP300 in solution-based assays

These applications enable researchers to investigate EP300 phosphorylation status across various experimental conditions, disease states, and tissue types, contributing to understanding EP300's role in cellular processes and pathological conditions .

How can I verify the specificity of a Phospho-EP300 (S89) antibody?

Verifying antibody specificity is crucial for reliable experimental outcomes. For Phospho-EP300 (S89) antibodies, you should:

• Perform dot blot analysis: Compare reactivity between phospho-peptide and non-phospho-peptide controls. Published data shows specific binding to phosphorylated target peptides at concentrations around 0.5 μg/ml

• Include phosphatase treatment controls: Samples treated with phosphatases should show reduced or eliminated signal if the antibody is truly phospho-specific

• Use genetic controls: Samples from EP300 knockout systems or S89A mutants (serine replaced with alanine to prevent phosphorylation) should show negative results

• Cross-validate with other detection methods: Mass spectrometry or alternative phospho-specific antibodies targeting different epitopes can confirm phosphorylation status

This multi-faceted approach ensures that observed signals genuinely represent S89 phosphorylation rather than non-specific binding or cross-reactivity with similar epitopes .

How does EP300 S89 phosphorylation status change in response to hypoxia and what experimental design would best capture this dynamic?

EP300 functions as a co-activator of HIF1A, a critical transcription factor in hypoxic response . To effectively study S89 phosphorylation dynamics under hypoxia:

• Time-course experiment design:

  • Expose cells to hypoxic conditions (1% O₂) for intervals (0, 1, 3, 6, 12, 24 hours)

  • Include both quick-onset and sustained hypoxia models

  • Compare with chemical hypoxia mimetics (CoCl₂, DMOG, DFO)

• Combined analytical approaches:

  • Quantitative Western blotting with Phospho-EP300 (S89) antibody

  • Immunoprecipitation followed by phospho-specific detection

  • Parallel immunofluorescence to assess subcellular localization changes

• Functional correlation:

  • Monitor HIF1A target gene expression (e.g., VEGF) simultaneously

  • Assess EP300 HAT activity in relation to phosphorylation state

  • Use S89A and S89D/E (phosphomimetic) mutants to confirm functional impact

This comprehensive approach allows for temporal resolution of phosphorylation events while connecting them to functional outcomes in hypoxic response pathways .

What are the key considerations when using Phospho-EP300 (S89) antibody for cancer tissue analysis?

When analyzing cancer tissues with Phospho-EP300 (S89) antibody, consider these critical factors:

• Tissue handling and fixation:

  • Phosphorylation modifications are labile; minimize time between tissue acquisition and fixation

  • Document fixation conditions precisely as overfixation can mask epitopes

  • Consider phosphatase inhibitor inclusion during sample preparation

• Validation in specific cancer contexts:

  • Experimental evidence has demonstrated successful application in breast carcinoma and hepatocarcinoma tissues

  • Each cancer type may require protocol optimization based on tissue architecture and composition

• Signal interpretation framework:

  • Establish scoring systems that account for:

    • Intensity of phospho-signal

    • Percentage of positive cells

    • Subcellular localization patterns (nuclear vs. cytoplasmic)

    • Heterogeneity within tumor regions

• Essential controls:

  Control Type	Purpose	Implementation
  Negative	Assess background/non-specific binding	IgG isotype control and/or phosphopeptide competition
  Positive	Confirm detection system functionality	Known positive sample (e.g., stimulated cell line lysate)
  Biological	Validate biological relevance	Compare with normal adjacent tissue
  Technical	Account for staining variability	Include reference tissues in each batch

This methodical approach enhances reliability when interpreting phosphorylation status in complex heterogeneous tissues like tumors .

How can Phospho-EP300 (S89) antibody be used to investigate the relationship between EP300 and transcriptional regulation?

To leverage this antibody for investigating EP300's role in transcriptional regulation:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Conduct sequential ChIP (first with Phospho-EP300 (S89) antibody, then with antibodies against interacting transcription factors)

  • Compare genome-wide binding patterns of total EP300 versus phosphorylated EP300(S89)

  • Correlate phosphorylation status with active transcription markers (H3K27ac, RNA Pol II)

• Functional transcriptional assays:

  • Reporter gene assays comparing wild-type EP300 vs. S89A mutant effects

  • RNA-seq following modulation of S89 phosphorylation status

  • Nascent transcription assays (e.g., PRO-seq) to capture immediate transcriptional impacts

• Protein interaction studies:

  • Compare interactome of phosphorylated vs. non-phosphorylated EP300

  • Assess how S89 phosphorylation affects interaction with CREB and other transcription factors

  • Investigate phosphorylation-dependent alterations in transcriptional complex assembly

These approaches connect the static detection of phosphorylation with dynamic functional outcomes in transcriptional regulation .

What protocol optimizations are recommended for immunohistochemistry using Phospho-EP300 (S89) antibody?

For optimal IHC results with Phospho-EP300 (S89) antibody:

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval methods (citrate pH 6.0 vs. EDTA pH 9.0)

  • Evaluate retrieval duration (10-30 minutes) for optimal signal-to-noise ratio

  • Consider dual pH approach if detecting multiple targets

• Antibody incubation parameters:

  • Recommended working dilution range: Typically start at 0.5-1 μg/ml (approximately 1:450-1:900 dilution from stock)

  • Incubation time-temperature combinations:

    • Overnight at 4°C (preferred for phospho-epitopes)

    • 1-2 hours at room temperature (if signal strength is adequate)

• Signal development system selection:

  • Peroxidase-based detection with AEC has demonstrated success in published applications

  • Consider tyramide signal amplification for low-abundance targets

  • Multiplexed detection may require fluorescence-based approaches

• Counterstaining considerations:

  • Light hematoxylin counterstaining preserves phospho-signal visibility

  • DAPI for nuclear definition in fluorescence applications

  • Avoid prolonged exposure to aqueous solutions which may leach phospho-epitopes

These methodological refinements help overcome common challenges in phospho-epitope detection while maximizing signal specificity and intensity .

What are the best practices for storage and handling of Phospho-EP300 (S89) antibody to maintain its activity?

To preserve antibody performance over time:

• Storage recommendations:

  • Store at -20°C to -80°C for long-term preservation

  • Avoid repeated freeze-thaw cycles which can degrade antibody quality

  • For working stocks, small aliquots (10-20 μl) minimize freeze-thaw damage

• Working solution preparation:

  • Dilute in fresh buffer containing suitable protein carrier (0.5% BSA)

  • For phospho-specific antibodies, consider phosphatase inhibitor addition to dilution buffers

  • Prepare working solutions immediately before use when possible

• Shipping and temporary storage:

  • Products typically ship with ice packs

  • Can be maintained at 2-8°C for short-term use (up to 1 week)

  • Return to -20°C promptly when not in active use

• Performance monitoring:

  • Periodically validate activity against known positive controls

  • Document lot-to-lot variations if observed

  • Expected shelf life is approximately 12 months when stored properly

These practices maximize antibody lifespan and experimental reproducibility while minimizing resource waste .

How can I troubleshoot weak or absent signal when using Phospho-EP300 (S89) antibody?

When encountering detection problems:

• Sample preparation assessment:

  • Phosphorylation modifications can be rapidly lost; evaluate sample handling time

  • Confirm phosphatase inhibitor inclusion in all buffers

  • Consider alternative fixation protocols if using histological specimens

• Technical optimization approach:

  • Titrate antibody concentration (try 2-3× higher concentration)

  • Extend primary antibody incubation time or adjust temperature

  • Evaluate different detection systems (HRP vs. fluorescence)

  • Employ signal amplification techniques (biotin-streptavidin, tyramide)

• Biological considerations:

  • Verify experimental conditions that promote S89 phosphorylation

  • Consider stimulus duration and strength (phosphorylation may be transient)

  • Use positive control samples with known phosphorylation status

• Antibody validation:

  • Perform dot blot with phosphopeptide control to confirm antibody activity

  • Compare multiple antibody lots if available

  • Consider alternative phospho-EP300 (S89) antibodies from different suppliers

This systematic troubleshooting framework addresses both technical and biological factors that may contribute to detection challenges .

How can I design experiments to investigate the kinases responsible for EP300 S89 phosphorylation?

To identify and validate the kinases that phosphorylate EP300 at S89:

• Kinase prediction and screening:

  • Perform in silico analysis of S89 sequence context using kinase prediction tools

  • Screen candidate kinases using in vitro kinase assays with recombinant EP300 fragments

  • Conduct kinase inhibitor panels to identify relevant pathways

• Cellular validation approaches:

  • Implement genetic approaches (siRNA/shRNA/CRISPR) targeting candidate kinases

  • Use phospho-specific antibody to monitor S89 phosphorylation changes

  • Create phosphorylation sensor constructs for live-cell imaging

• Physical interaction demonstration:

  • Co-immunoprecipitation of EP300 with candidate kinases

  • Proximity ligation assays to verify interactions in intact cells

  • Mass spectrometry-based approaches to identify kinase-substrate complexes

• Functional validation:

  • Determine how kinase inhibition affects EP300-dependent transcriptional events

  • Assess whether kinase overexpression enhances S89 phosphorylation

  • Evaluate physiological stimuli that activate both the kinase and increase S89 phosphorylation

This comprehensive approach provides multiple lines of evidence for kinase identification and validation .

What comparative analysis can be performed between total EP300 and Phospho-EP300 (S89) in research settings?

Effective comparative analyses include:

• Ratio-based approaches:

  • Calculate phospho-to-total EP300 ratios across experimental conditions

  • Normalize phospho-signals to total protein to distinguish regulation mechanisms

  • Track temporal changes in phosphorylation ratio during cellular processes

• Localization comparison methods:

  • Perform dual immunofluorescence with total and phospho-specific antibodies

  • Conduct subcellular fractionation followed by Western blot analysis

  • Use proximity ligation assays to identify differential interaction partners

• Functional comparative assays:

  • ChIP-seq with both antibodies to identify phosphorylation-dependent genomic binding sites

  • Assess differential protein complex formation using proteomics approaches

  • Compare transcriptional activation potential between phosphorylated and total pools

• Differential stimulus response:

  • Create response profiles across stressors (hypoxia, DNA damage, inflammation)

  • Develop phosphorylation kinetics models under various conditions

  • Identify conditions where total protein and phospho-form are differentially regulated

These comparative analyses provide deeper insights into how S89 phosphorylation modulates EP300 function in different biological contexts .

How can Phospho-EP300 (S89) antibody be integrated into multi-parameter analysis workflows?

For comprehensive multi-parameter studies:

• Multiplexed imaging approaches:

  • Combine with other phospho-specific and total protein antibodies using:

    • Sequential immunofluorescence with spectral unmixing

    • Multiplexed immunohistochemistry with chromogenic detection

    • Cyclic immunofluorescence for high-parameter tissue analysis

• Flow cytometry and mass cytometry applications:

  • Develop intracellular staining protocols for phospho-EP300 detection

  • Combine with cell surface markers and other intracellular targets

  • Create high-dimensional datasets for computational analysis

• Integration with omics technologies:

  • Correlate phospho-EP300 status with:

    • Transcriptome data (RNA-seq)

    • Chromatin accessibility (ATAC-seq)

    • Global phosphoproteome patterns

• Single-cell analysis integration:

  • Develop compatible fixation and permeabilization protocols

  • Combine with other signaling markers for pathway profiling

  • Create computational frameworks for analyzing heterogeneous responses

This integration enables contextual understanding of EP300 phosphorylation within broader cellular signaling networks and phenotypic outcomes .

What are the critical quality control parameters for validating Phospho-EP300 (S89) antibody performance?

Essential quality control measures include:

• Specificity verification:

  • Dot blot comparison between phospho and non-phospho peptides (50 ng per dot)

  • Signal elimination through phosphopeptide competition

  • Absence of signal in S89A mutant samples

• Sensitivity assessment:

  • Determine limit of detection using serial dilutions of positive control

  • Evaluate signal-to-noise ratio across applications

  • Compare with other phospho-EP300 detection methods

• Application-specific validation:

  • For IHC: Verify consistent staining patterns in known positive tissues

  • For dot blot: Confirm detection at recommended working concentration (0.5 μg/ml)

  • For ELISA: Establish standard curves with recombinant phosphorylated protein

• Batch consistency verification:

  • Compare lot-to-lot performance using standardized samples

  • Document any variations in optimal working concentrations

  • Maintain reference standards for long-term studies

These validation steps ensure reliable performance across experimental applications and minimize variability in research outcomes .

What are the comparative advantages of using rabbit polyclonal Phospho-EP300 (S89) antibodies versus other antibody formats?

The advantages of rabbit polyclonal antibodies for phospho-EP300 detection include:

• Epitope recognition characteristics:

  • Polyclonal nature provides recognition of multiple epitopes surrounding the S89 site

  • Enhanced signal through multiple binding events per target molecule

  • Greater tolerance to minor conformational changes or partial epitope masking

• Production considerations:

  • Generated using KLH-conjugated synthetic phosphopeptides

  • Affinity purified through protein A columns followed by peptide affinity purification

  • Rabbit host provides typically higher affinity than mouse-derived antibodies

• Application versatility:

  • Effectively functions across multiple applications (IHC-P, dot blot, ELISA)

  • Works well in formalin-fixed paraffin-embedded tissues after appropriate retrieval

  • Compatible with multiple detection systems (enzymatic and fluorescent)

• Limitations and alternatives:

  • Lot-to-lot variation may require standardization

  • For absolute quantification, consider phospho-specific monoclonal antibodies

  • For super-resolution imaging, smaller detection agents (Fabs, nanobodies) may be preferable