EP300 Antibody, Biotin conjugated

What is EP300 and why is it significant in epigenetic research?

EP300 (E1A Binding Protein P300) is a critical histone acetyltransferase that functions as a transcriptional coactivator regulating gene expression through chromatin remodeling. It plays a fundamental role in establishing H3K27ac at essential gene enhancers, particularly in high-risk pediatric cancers such as neuroblastoma. EP300's significance stems from its ability to acetylate histones and non-histone proteins, thereby modulating chromatin accessibility and transcriptional activity. Research has demonstrated that EP300 is recruited to DNA through interactions with specific transcription factors, including TFAP2β, which is part of the lineage-defining core regulatory circuitry in neuroblastoma cells. Understanding EP300's function is crucial for elucidating epigenetic mechanisms controlling gene expression in both normal development and disease states .

What are the main applications of EP300 Antibody, Biotin conjugated in research?

EP300 Antibody, Biotin conjugated serves multiple research applications, with the primary ones being Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), and Dot Blot (DB) . The biotin conjugation significantly enhances detection sensitivity through streptavidin-based systems, making this antibody particularly valuable for studying low-abundance EP300 protein. In immunohistochemistry applications, the biotin-conjugated antibody enables visualization of EP300 localization in tissue sections, while in Western blotting, it allows for specific detection of phosphorylated EP300 protein. This antibody can also be employed in chromatin immunoprecipitation (ChIP) assays to investigate EP300 binding to specific genomic regions, although optimization may be required depending on the experimental system .

How does the phosphorylation state of EP300 affect its functional properties?

The phosphorylation state of EP300 serves as a critical regulatory mechanism that modulates its acetyltransferase activity, protein-protein interactions, and subcellular localization. The Rabbit Anti-EP300 phosphorylated Antibody specifically recognizes EP300 phosphorylated at Ser89 , which represents an important regulatory site. Phosphorylation at specific residues can either enhance or inhibit EP300's acetyltransferase activity, affecting its ability to acetylate histones and establish active enhancers. In neuroblastoma cells, phosphorylated EP300 demonstrates preferential binding to certain transcription factors like TFAP2β compared to its non-phosphorylated state. Researchers studying EP300 function should consider using phospho-specific antibodies like the Biotin-conjugated version to distinguish between different functional states of EP300 and correlate these states with specific cellular processes or disease conditions .

What is the difference between EP300 and CBP in terms of chromatin binding patterns?

Despite their structural similarities, EP300 and CBP (CREB-binding protein) exhibit distinct genome-wide binding patterns and functional roles. Research in neuroblastoma cell lines has demonstrated that while the majority of sites genome-wide bound by EP300 are also bound by CBP, a small number of sites are preferentially bound by either EP300 or CBP . Notably, EP300 shows preferential localization at sites controlling the expression of adrenergic core regulatory circuitry genes in neuroblastoma cells, where CBP is minimally enriched. This selective binding pattern is partly explained by EP300's physical interaction with specific transcription factors like TFAP2β and GATA3, interactions not observed with CBP. These differences highlight the non-redundant roles of these two acetyltransferases and underscore the importance of using specific antibodies when studying their distinct functions in different cellular contexts .

How does biotin conjugation benefit EP300 antibody detection systems?

Biotin conjugation to EP300 antibodies provides several methodological advantages in research applications. The high-affinity interaction between biotin and streptavidin (Kd ≈ 10^-15 M) offers exceptional sensitivity and specificity in detection systems. This conjugation allows for signal amplification through the use of streptavidin-coupled detection reagents, enhancing the detection limit of low-abundance EP300 protein in complex biological samples. Additionally, biotin-conjugated antibodies provide flexibility in experimental design, as they can be used with various detection systems including streptavidin-HRP for Western blotting, streptavidin-fluorophores for immunofluorescence, or streptavidin-coupled magnetic beads for immunoprecipitation procedures. The Biotin-conjugated EP300 antibody is supplied as a liquid in PBS (pH 7.2) without preservatives, optimizing its use in sensitive applications where additives might interfere with experimental outcomes .

How can researchers optimize EP300 Antibody, Biotin conjugated for chromatin immunoprecipitation (ChIP) assays?

Optimizing EP300 Antibody, Biotin conjugated for ChIP assays requires several methodological considerations. Begin by performing antibody titration experiments (1:100 to 1:1000 dilutions) to determine the optimal concentration that maximizes signal-to-noise ratio. Since the antibody recognizes phosphorylated EP300 at Ser89 , ensure your chromatin preparation protocol preserves phosphorylation states by including phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in all buffers. For biotin-conjugated antibodies, use streptavidin-coupled magnetic beads rather than Protein A/G beads for immunoprecipitation. Implement a pre-clearing step with unconjugated streptavidin beads to reduce non-specific binding. Additionally, include biotin blocking steps to prevent non-specific interactions between endogenous biotinylated proteins and streptavidin. Validate your ChIP-seq results by performing parallel experiments with antibodies recognizing different EP300 epitopes and compare binding patterns with H3K27ac ChIP-seq data, as EP300 is known to establish H3K27ac at enhancers in neuroblastoma and other cell types .

What methodological considerations should be taken when using EP300 Antibody, Biotin conjugated across different cell types?

When employing EP300 Antibody, Biotin conjugated across different cell types, researchers must address several methodological variables. First, expression levels of EP300 vary significantly between cell types, necessitating optimization of antibody concentration for each system. The phosphorylation state of EP300 at Ser89 also differs across cell lineages and cellular conditions, affecting antibody recognition. For neuroblastoma studies, consider the MYCN amplification status of your cell lines, as this impacts EP300 function and expression . For cell types with high endogenous biotin or biotinylated proteins (e.g., liver cells), implement additional blocking steps using free biotin or avidin to reduce background.

Extraction protocols should be tailored to different cell types—neuronal cells often require gentler lysis conditions than fibroblasts. Finally, validate antibody specificity in each new cell type using EP300 knockdown or knockout controls. Western blot analysis comparing EP300 detection in nuclear extracts from different cell types can help establish optimal working conditions and confirm the antibody's cross-reactivity with your specific experimental system .

How can researchers validate the specificity of EP300 Antibody, Biotin conjugated in their experimental systems?

Validating the specificity of EP300 Antibody, Biotin conjugated requires a multi-faceted approach. Begin with Western blot analysis using positive controls (cells known to express EP300) and negative controls (EP300 knockout/knockdown cells) to confirm the antibody detects a single band at the expected molecular weight (~300 kDa). Perform peptide competition assays using the immunizing phosphopeptide (corresponding to amino acid residues surrounding Ser89 of human EP300) to demonstrate signal reduction when the antibody is pre-incubated with the target epitope. For phospho-specific validation, treat lysates with lambda phosphatase and confirm signal loss.

In immunoprecipitation experiments, validate specificity by mass spectrometry analysis of immunoprecipitated proteins to confirm EP300 enrichment. For ChIP applications, compare binding profiles with published EP300 ChIP-seq datasets and confirm co-localization with H3K27ac marks at enhancers . Additionally, perform parallel experiments with alternative EP300 antibodies targeting different epitopes and compare results. This comprehensive validation strategy ensures reliable and reproducible results when working with this biotin-conjugated antibody in diverse research applications.

What technical challenges arise when studying EP300 phosphorylation using phospho-specific antibodies?

Studying EP300 phosphorylation presents several technical challenges that researchers must address methodically. First, phosphorylation is a transient, dynamic modification that can be rapidly lost during sample preparation—implement immediate cell lysis in buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails) to preserve phosphorylation states. The large size of EP300 (~300 kDa) complicates efficient protein transfer in Western blotting; use low percentage gels (6-8%) and extended transfer times with specialized buffers for high molecular weight proteins.

The phospho-specific EP300 antibody recognizes phosphorylation at Ser89 , but EP300 contains multiple phosphorylation sites that may exhibit crosstalk, requiring complementary approaches such as mass spectrometry to comprehensively characterize phosphorylation patterns. Background signal can be problematic in immunostaining applications; optimize blocking conditions (consider 5% BSA rather than milk, which contains phosphoproteins) and include dephosphorylated controls. Finally, context-dependent phosphorylation varies with cell type, growth conditions, and signaling states, necessitating carefully timed experiments and appropriate stimulation protocols when studying signaling-dependent EP300 phosphorylation events.

How can EP300 Antibody, Biotin conjugated be used to investigate EP300's role in enhancer regulation?

Investigating EP300's role in enhancer regulation requires integrative approaches utilizing EP300 Antibody, Biotin conjugated. Begin with ChIP-seq experiments to map genome-wide EP300 binding sites, particularly focusing on enhancer regions marked by H3K27ac. The biotin conjugation facilitates efficient immunoprecipitation using streptavidin magnetic beads, allowing for high sensitivity and specificity in ChIP-seq applications. Combine EP300 ChIP-seq with transcription factor ChIP-seq (particularly TFAP2β and GATA3 in neuroblastoma models) to identify co-localization patterns and potential recruitment mechanisms.

Implement CUT&RUN or CUT&Tag as complementary approaches for higher resolution EP300 binding profiles. For functional validation, couple EP300 degradation experiments using PROTAC agents like JQAD1 with enhancer activity assays (luciferase reporters) and RNA-seq to correlate EP300 loss with changes in enhancer activity and gene expression. Employ chromosome conformation capture techniques (4C, Hi-C) to examine how EP300 binding affects three-dimensional chromatin architecture. Time-course experiments following EP300 degradation or inhibition can reveal the dynamics of enhancer decommissioning, providing insights into the temporal requirements for EP300 in maintaining enhancer activity across different genomic contexts.

What controls should be included when studying EP300 interactions with transcription factors?

Additional methodological controls should include testing the interaction in multiple cell types to establish biological relevance, performing binding assays with deletion mutants to map interaction domains, and validating protein-protein interactions through orthogonal methods such as proximity ligation assays or FRET microscopy. For studying phosphorylation-dependent interactions, compare results using phospho-specific and pan-EP300 antibodies under various cellular conditions that modulate phosphorylation status .

How can researchers differentiate between EP300 and CBP binding in experimental systems?

Differentiating between EP300 and CBP binding requires strategic experimental approaches due to their structural similarities. First, employ highly specific antibodies—the phospho-specific EP300 antibody (Biotin conjugated) recognizing Ser89 provides good specificity as CBP lacks an equivalent phosphorylation site. In ChIP-seq experiments, perform sequential ChIP (re-ChIP) to identify genomic regions bound exclusively by EP300 versus regions bound by both factors. Implement CRISPR-Cas9 knockout of either EP300 or CBP followed by ChIP-seq of the remaining factor to identify sites with redundant versus specific binding.

For protein-protein interaction studies, use stringent immunoprecipitation conditions that maintain specific interactions while reducing non-specific binding. Mass spectrometry analysis of immunoprecipitated complexes can identify unique interaction partners that distinguish EP300 from CBP complexes. Research in neuroblastoma cells has demonstrated that EP300, but not CBP, physically interacts with transcription factors like TFAP2β , providing a functional readout to distinguish these factors. Finally, use selective degradation approaches with PROTAC agents like JQAD1, which preferentially degrades EP300 over CBP , to dissect factor-specific functions through temporal depletion followed by functional or genomic assays.

What approaches can be used to study EP300 degradation in combination with PROTAC agents?

Studying EP300 degradation using PROTAC agents requires systematic experimental approaches that leverage the biotin-conjugated EP300 antibody. Begin with time-course experiments monitoring EP300 protein levels via Western blot after PROTAC treatment (e.g., JQAD1) , using the biotin-conjugated antibody for enhanced detection sensitivity. Quantify degradation kinetics by densitometry analysis normalized to loading controls, establishing the optimal time points for subsequent functional studies. Implement cycloheximide chase assays in the presence and absence of the PROTAC to differentiate between degradation and inhibited protein synthesis.

To confirm the mechanism of action, pre-treat cells with proteasome inhibitors (MG132) or neddylation inhibitors (MLN4924) before adding the PROTAC, which should block EP300 degradation if proteasome-dependent. Use confocal microscopy with fluorescently labeled streptavidin to visualize changes in EP300 localization during degradation. For target validation, perform PROTAC-biotin pulldowns followed by Western blotting to confirm ternary complex formation between EP300, the PROTAC, and E3 ligase components like CRBN . Couple degradation studies with functional readouts such as H3K27ac ChIP-seq, ATAC-seq, and RNA-seq to correlate the temporal loss of EP300 with changes in chromatin accessibility, histone modifications, and gene expression, particularly at enhancer regions controlling core regulatory circuitry in cancer cells .

How can researchers troubleshoot inconsistent results when using EP300 Antibody, Biotin conjugated?

Troubleshooting inconsistent results with EP300 Antibody, Biotin conjugated requires systematic assessment of multiple experimental variables. First, evaluate antibody stability—store aliquots at -20°C and avoid repeated freeze-thaw cycles that can compromise binding activity. Verify the phosphorylation state is preserved during sample preparation by comparing fresh lysates with stored samples using Western blot. For immunoprecipitation applications, test different lysis buffers (RIPA vs. NP-40 vs. Digitonin-based) as buffer composition significantly affects epitope accessibility, particularly for phosphorylated residues .

When inconsistencies occur in immunohistochemistry, optimize antigen retrieval methods (citrate vs. EDTA-based) and fixation protocols (paraformaldehyde concentration and duration). For biotin-conjugated antibodies specifically, implement additional blocking steps with avidin/biotin blocking kits to reduce endogenous biotin interference. Consider batch effects in streptavidin reagents by testing multiple lots. Cell-type specific factors may also contribute to variability—verify EP300 expression levels and phosphorylation status across experimental systems. Finally, compare results using alternative EP300 antibodies targeting different epitopes to distinguish antibody-specific issues from biological variability. Document all optimization parameters in a controlled manner to establish reproducible protocols for your specific experimental system .

What considerations should guide experimental design when studying EP300's role in specific cancer types?

When designing experiments to study EP300's role in cancer using the biotin-conjugated antibody, researchers should implement a comprehensive strategic approach. First, select appropriate cellular models—for neuroblastoma studies, consider both MYCN-amplified (BE2C, Kelly) and non-amplified cell lines to account for MYCN-dependent effects on EP300 function . Compare EP300 binding patterns in cancer cells versus relevant non-transformed cells to identify cancer-specific EP300 genomic targeting. Integrate multi-omics approaches by combining EP300 ChIP-seq with H3K27ac ChIP-seq, ATAC-seq, and RNA-seq to correlate EP300 binding with enhancer activity and gene expression.

For functional studies, employ both genetic (CRISPR, shRNA) and chemical (JQAD1 PROTAC) approaches to modulate EP300 activity, as these provide complementary insights into acute versus chronic EP300 loss . Examine context-dependent interactions with lineage-specific transcription factors (e.g., TFAP2β in neuroblastoma) through co-immunoprecipitation and sequential ChIP. Consider the impact of tumor microenvironment by studying EP300 under hypoxia, nutrient deprivation, or co-culture conditions. For translational relevance, correlate experimental findings with patient sample analyses using tissue microarrays stained with the biotin-conjugated EP300 antibody, connecting molecular mechanisms to clinical outcomes and potential therapeutic vulnerabilities in specific cancer subtypes.

How should researchers analyze EP300 binding patterns in ChIP-seq experiments?

Analyzing EP300 binding patterns in ChIP-seq experiments requires a systematic bioinformatic approach tailored to enhancer biology. Begin with standard quality control metrics (ENCODE guidelines) including library complexity, fragment size distribution, and enrichment of signal over background. For peak calling, use algorithms sensitive to broad peaks (MACS2 with appropriate parameters) as EP300 often binds to extended enhancer regions rather than focal sites. Implement differential binding analysis when comparing EP300 occupancy across conditions, using packages like DiffBind or DESeq2 applied to normalized read counts in peak regions.

For biological interpretation, perform motif enrichment analysis to identify transcription factor binding sites enriched under EP300 peaks, which can reveal recruitment mechanisms similar to the TFAP2β and GATA3 motifs identified in neuroblastoma cells . Integrate H3K27ac ChIP-seq data to distinguish active from poised enhancers and classify super-enhancers using ranking of enhancer domains by signal intensity. Analyze co-localization with lineage-specific transcription factors through genomic intersection analysis and generate aggregate plots centered on transcription factor peaks. Finally, correlate EP300 binding with gene expression data by assigning enhancers to target genes through nearest-gene approaches or chromosome conformation data, enabling identification of EP300-dependent transcriptional programs in your biological system .

What statistical methods are appropriate for analyzing EP300 phosphorylation levels across experimental conditions?

Analyzing EP300 phosphorylation levels across experimental conditions requires appropriate statistical methods that account for the unique characteristics of phosphoprotein data. For Western blot quantification using the phospho-specific EP300 antibody , implement densitometry analysis with normalization to both total EP300 and a loading control (GAPDH, β-actin). Apply log transformation to densitometry data to achieve more normal distribution before statistical testing. For comparing multiple conditions, use one-way ANOVA followed by post-hoc tests (Tukey's or Dunnett's) with correction for multiple comparisons.

For more quantitative approaches like ELISA or mass spectrometry-based phosphoproteomics, implement robust linear models that account for batch effects and technical variability. Consider employing paired statistical tests when analyzing the same samples before and after treatments affecting phosphorylation. For time-course experiments monitoring phosphorylation dynamics, apply repeated measures ANOVA or mixed-effects models that account for within-subject correlation. Visualize phosphorylation data using box plots with individual data points or violin plots to represent data distribution. When correlating phosphorylation levels with functional outcomes, use regression analysis with appropriate transformations and outlier detection. Finally, calculate confidence intervals around fold changes to better represent the uncertainty in phosphorylation measurements across experimental conditions.

How can researchers integrate EP300 binding data with other epigenomic datasets?

Integrating EP300 binding data with other epigenomic datasets enables comprehensive understanding of enhancer regulation and transcriptional control mechanisms. Begin with correlation analysis between EP300 and histone modification ChIP-seq signals (particularly H3K27ac, H3K4me1, and H3K4me3) to classify active enhancers versus promoters. Implement genome segmentation approaches using tools like ChromHMM or Segway to define chromatin states based on combinations of EP300 and other epigenetic marks. Generate metaplots and heatmaps centered on EP300 peaks to visualize the distribution of various histone marks and chromatin accessibility (ATAC-seq) in relation to EP300 binding sites.

For more sophisticated integration, employ multivariate statistical methods such as principal component analysis or t-SNE to identify patterns in high-dimensional epigenomic data. Construct enhancer networks by connecting EP300-bound enhancers to target genes using chromosome conformation data (Hi-C, 4C) and correlate these networks with transcriptional outputs from RNA-seq data. Perform integrative analysis of EP300 binding with transcription factor ChIP-seq data (particularly TFAP2β and GATA3 in neuroblastoma contexts) to identify cooperative binding and potential enhanceosome formation. Finally, apply machine learning approaches to predict enhancer activity based on combinations of EP300 binding and other epigenetic features, enabling the identification of critical parameters determining enhancer functionality in specific cellular contexts .

What controls and validation methods should be used when interpreting co-immunoprecipitation results with EP300 Antibody, Biotin conjugated?

For biological validation, confirm interactions through reciprocal co-IP where the suspected binding partner (e.g., TFAP2β) is immunoprecipitated and EP300 is detected by Western blot. Employ domain mapping through truncation mutants or peptide competition to identify specific interaction interfaces. For interactions detected in cell lines, validate physiological relevance in primary tissues or through genetic manipulation of the interacting partners. Quantify interaction strength through densitometry analysis of Western blots, normalizing co-IP signal to input and IP efficiency. Consider additional orthogonal methods such as proximity ligation assay, FRET, or split-luciferase complementation to confirm interactions in intact cells. Finally, for phosphorylation-dependent interactions, compare results using the phospho-specific EP300 antibody versus pan-EP300 antibodies under conditions that modulate the phosphorylation state .

How can researchers quantitatively compare EP300 degradation efficiency across different PROTAC compounds?

Quantitatively comparing EP300 degradation efficiency across different PROTAC compounds requires standardized methodologies and appropriate metrics. Begin by establishing dose-response relationships for each compound through Western blot analysis using the biotin-conjugated EP300 antibody , treating cells with a concentration range (typically 0.1-10 μM) for a fixed time point (12-24 hours). Calculate DC50 values (concentration causing 50% degradation) through four-parameter logistic regression of densitometry data. Determine Dmax (maximum degradation achieved) to assess compound efficacy.

For kinetic analysis, perform time-course experiments (0-48 hours) at fixed PROTAC concentrations near the DC50, calculating degradation half-lives through non-linear regression. Compare hook effects (reduced efficacy at high concentrations) across compounds by testing extended concentration ranges. Assess selectivity by simultaneously monitoring CBP levels, calculating EP300/CBP degradation ratios to quantify specificity . For mechanistic comparisons, measure ternary complex formation efficiency through cellular thermal shift assays or bioluminescence resonance energy transfer. Evaluate the functional impact of degradation by correlating EP300 protein loss with reduction in H3K27ac levels through parallel Western blots or ChIP-qPCR at known EP300 target enhancers.

Finally, compare wash-out recovery kinetics to assess compound residence time and degradation persistence. These comprehensive metrics enable detailed comparison of PROTAC compounds beyond simple potency measures, providing insights into their mechanistic differences and potential therapeutic applications in EP300-dependent disease contexts .

What is the optimal protocol for using EP300 Antibody, Biotin conjugated in multiplex immunofluorescence studies?

The optimal protocol for multiplex immunofluorescence with EP300 Antibody, Biotin conjugated requires careful optimization to achieve specific detection while minimizing background. Begin with sample preparation: fix cells with 4% paraformaldehyde (10 minutes at room temperature) or tissues using standard formalin fixation, followed by antigen retrieval in citrate buffer (pH 6.0) for 20 minutes at 95°C. Block with 5% BSA containing avidin (10 μg/ml) for 1 hour to mask endogenous biotin, followed by biotin blocking (50 μg/ml) for 30 minutes.

For multiplex staining, apply the biotin-conjugated EP300 antibody at optimized dilution (typically starting at 1:100-1:500) overnight at 4°C . After washing, detect with fluorophore-conjugated streptavidin (Alexa Fluor 488 or 594) for 1 hour at room temperature. Before applying additional primary antibodies, implement an additional blocking step using unconjugated Fab fragments against the host species of the first antibody. For nuclear antigens like EP300, include DAPI counterstaining to facilitate nuclear localization assessment.

When combining with phospho-specific markers, maintain phosphatase inhibitors throughout the protocol. For tyramide signal amplification to enhance detection sensitivity, use streptavidin-HRP followed by tyramide-fluorophore reaction, then perform heat-mediated stripping before subsequent antibody applications. Validate multiplex protocol through single-color controls and fluorophore minus one (FMO) controls to ensure specific detection without spectral overlap or antibody cross-reactivity.

How can researchers adapt EP300 Antibody, Biotin conjugated for high-throughput screening applications?

Adapting EP300 Antibody, Biotin conjugated for high-throughput screening requires methodological modifications to ensure reproducibility across large sample sets. Begin by optimizing antibody concentration in a miniaturized format—perform checkerboard titrations in 384-well plates to determine the minimum concentration providing reliable signal-to-background ratios. For cell-based screening, establish automated fixation and immunostaining protocols using robotic liquid handlers, standardizing incubation times and washing steps to minimize well-to-well variability.

Implement in-plate controls: positive controls (cells with known high EP300 phosphorylation), negative controls (EP300 knockout cells or phosphatase-treated samples), and technical controls (secondary-only wells). For detection, utilize high-content imaging systems with streptavidin-fluorophore detection or develop an ELISA-based approach using streptavidin-HRP and chemiluminescent substrates for plate reader detection. Calculate Z'-factors for each plate to assess assay quality (Z' > 0.5 indicates an excellent assay window).

For screening applications targeting EP300 degradation (e.g., novel PROTAC compounds) , develop a homogeneous assay format using proximity-based detection such as AlphaLISA with biotinylated EP300 antibody and a second antibody against total EP300. This approach enables real-time monitoring of EP300 levels without washing steps, increasing throughput. Finally, implement data normalization strategies to account for plate-to-plate variation, cell number differences, and edge effects, ensuring robust analysis of EP300 levels across large compound or genetic perturbation libraries.

What methodological approaches enable simultaneous detection of EP300 phosphorylation and protein-protein interactions?

Simultaneous detection of EP300 phosphorylation and protein-protein interactions requires sophisticated methodological approaches that integrate multiple techniques. One effective strategy employs sequential or dual immunoprecipitation: first immunoprecipitate with the biotin-conjugated phospho-specific EP300 antibody using streptavidin beads, then elute under mild conditions (biotin competition rather than denaturing elution) and perform a second immunoprecipitation targeting the interacting protein (e.g., TFAP2β) . This approach enriches for complexes containing phosphorylated EP300 specifically engaged in the interaction of interest.

Alternatively, implement proximity ligation assay (PLA) using the biotin-conjugated phospho-EP300 antibody with streptavidin-oligonucleotide conjugates alongside antibodies against potential interacting partners. This technique generates fluorescent signals only when proteins are within 40 nm proximity, enabling visualization of specific phospho-EP300 interaction complexes in situ. For higher-throughput approaches, develop a BiFC (Bimolecular Fluorescence Complementation) system where EP300 is fused to one half of a split fluorescent protein and potential interaction partners to the complementary half, then use the phospho-specific antibody for immunoprecipitation of complexes showing fluorescence.

Mass spectrometry-based approaches offer comprehensive analysis: perform immunoprecipitation with the phospho-specific EP300 antibody followed by mass spectrometry to identify interacting proteins while simultaneously confirming the phosphorylation state through phosphopeptide analysis. This integrated approach provides a systems-level view of how EP300 phosphorylation modulates its protein interaction network in different cellular contexts .

How should researchers optimize blocking conditions when using EP300 Antibody, Biotin conjugated in different applications?

Optimizing blocking conditions for EP300 Antibody, Biotin conjugated applications requires tailored approaches addressing both general background and biotin-specific challenges. For Western blotting applications, compare blocking reagents systematically: 5% BSA in TBST typically outperforms milk-based blockers for phospho-specific antibodies like the phospho-EP300 (Ser89) antibody , as milk contains phosphoproteins that can compete for antibody binding. Implement a dual blocking approach—first block with 5% BSA for 1 hour, then add avidin (10 μg/ml) for an additional 30 minutes to sequester endogenous biotin, followed by biotin blocking (50 μg/ml) before antibody incubation.

For immunohistochemistry or immunofluorescence, extend blocking times (2 hours minimum) and include 0.3% Triton X-100 to enhance nuclear penetration for detecting EP300. Test commercial biotin blocking kits against homemade alternatives to determine optimal background reduction. Cell-type specific optimization is crucial—liver and kidney tissues contain high levels of endogenous biotin requiring more stringent blocking protocols.

For immunoprecipitation applications, pre-clear lysates with unconjugated streptavidin beads before adding the biotin-conjugated antibody to reduce non-specific binding. When troubleshooting high background, incorporate additional washing steps with increasing stringency (0.1% to 0.5% Tween-20 or NP-40). Finally, optimize antibody concentration through titration experiments for each application and cell type, as excess antibody increases non-specific binding while insufficient antibody reduces detection sensitivity .

What innovative applications can be developed by combining EP300 Antibody, Biotin conjugated with emerging genomic technologies?

Combining EP300 Antibody, Biotin conjugated with emerging genomic technologies creates innovative research applications that advance our understanding of enhancer biology. One promising approach integrates CUT&Tag technology with the biotin-conjugated antibody—the pre-existing biotin tag eliminates the need for secondary antibody incubation, streamlining the protocol while maintaining specificity for phosphorylated EP300 . This modified CUT&Tag approach enables high-resolution mapping of phospho-EP300 genomic binding with minimal cell numbers, ideal for rare cell populations or clinical samples.

Another innovative application combines the biotin-conjugated EP300 antibody with Proximity Ligation-Assisted ChIP-seq (PLAC-seq) to simultaneously identify EP300-bound enhancers and their physical interactions with target promoters. This approach reveals the functional enhancer-promoter relationships regulated by EP300 in three-dimensional nuclear space. For single-cell applications, adapt the antibody for use in scCUT&Tag or scCUT&RUN protocols to profile EP300 binding heterogeneity at single-cell resolution, revealing cell state-specific enhancer usage patterns.

Develop spatial transcriptomics applications by combining the biotin-conjugated antibody with in situ sequencing technologies, mapping EP300 localization in relation to active transcription within tissue architecture. Finally, implement live-cell imaging of EP300 dynamics by conjugating fluorescent streptavidin to the biotin-tagged antibody in permeabilized cells, enabling real-time visualization of EP300 recruitment to enhancers following signaling events. These innovative approaches leverage the specificity and biotin conjugation of the EP300 antibody to address fundamental questions about enhancer regulation in development and disease .

What are the emerging trends in EP300 research that might influence future applications of biotin-conjugated EP300 antibodies?

Emerging trends in EP300 research point to exciting future applications for biotin-conjugated EP300 antibodies. First, the development of selective PROTAC degraders like JQAD1 is advancing rapidly, creating opportunities to use biotin-conjugated EP300 antibodies in monitoring degradation kinetics and target selectivity in high-throughput screening platforms. The growing recognition of EP300's role in phase separation and biomolecular condensate formation suggests applications in studying how phosphorylation states affect EP300's participation in transcriptional condensates at super-enhancers.

Single-cell epigenomic technologies are evolving to incorporate antibody-based approaches, opening possibilities for adapting biotin-conjugated EP300 antibodies for single-cell CUT&Tag or single-cell CUT&RUN to profile enhancer usage heterogeneity at unprecedented resolution. The emerging field of epitranscriptomics reveals connections between enhancer activity and RNA modifications, suggesting novel applications combining EP300 ChIP with RNA immunoprecipitation to investigate enhancer RNA regulation.

Advances in cryo-electron microscopy are improving structural characterization of large protein complexes, potentially enabling visualization of EP300-containing enhanceosomes using biotin-conjugated antibodies for labeling and identification. Finally, the translation of EP300-targeted therapies to clinical applications will drive demand for companion diagnostic approaches using biotin-conjugated phospho-specific antibodies to stratify patients and monitor treatment efficacy in precision medicine applications for EP300-dependent cancers like neuroblastoma .

How will methodological advances influence the utility of EP300 Antibody, Biotin conjugated in future research?

Methodological advances will substantially enhance the utility of EP300 Antibody, Biotin conjugated in future research through several emerging technologies. Microfluidic antibody-based technologies are rapidly developing, enabling integration of the biotin-conjugated antibody into chip-based platforms for automated, high-throughput ChIP procedures requiring minimal sample input—critical for analyzing clinical specimens with limited material. Advanced microscopy techniques including super-resolution approaches (STORM, PALM) combined with biotin-streptavidin detection systems will provide unprecedented visualization of EP300 localization within nuclear architecture at nanometer resolution.

CRISPR-based technologies for in situ protein tagging can be combined with the biotin-conjugated antibody for dynamic tracking of EP300 in living cells, enabling real-time monitoring of enhancer assembly and disassembly. Mass cytometry (CyTOF) adaptations using metal-conjugated streptavidin will allow multiplexed detection of phospho-EP300 alongside dozens of other cellular markers in heterogeneous populations, providing deeper insights into EP300 regulation across different cell states.