Phospho-Histone H3 (Thr11) Antibody

What is Phospho-Histone H3 (Thr11) and why is it significant in cell biology?

Phospho-Histone H3 (Thr11) refers to the phosphorylated form of histone H3 at the threonine residue at position 11. This specific post-translational modification plays a crucial role during mitosis, particularly in chromosome condensation and segregation. Unlike other histone modifications, phosphorylation at Thr11 is largely restricted to centromeric regions of chromosomes and occurs during a specific window of mitosis from prophase to early anaphase. Dysregulation of this phosphorylation event has been associated with various diseases, including cancer, making it an important target for research in oncology and cell biology . The spatial and temporal specificity of this modification suggests it may have specialized functions in centromere biology distinct from other histone phosphorylation events .

How does Phospho-Histone H3 (Thr11) differ from other histone H3 modifications?

Phospho-Histone H3 (Thr11) differs from other histone H3 modifications in several key aspects. First, while Histone H3 phosphorylation at Ser10 is more abundant and distributed throughout chromosomes (particularly in distal regions), Thr11 phosphorylation is less abundant and predominantly localized to centromeric regions as revealed by confocal microscopy studies . Second, the temporal pattern differs: Ser10 phosphorylation appears earlier in late G2/early prophase and persists until anaphase, whereas Thr11 phosphorylation is more restricted to the window between prophase and early anaphase . Third, evidence suggests that Ser10 and Thr11 phosphorylations may be mutually exclusive, indicating potential regulatory interplay between these modifications . These distinctions suggest specialized roles for Thr11 phosphorylation in centromere function during mitosis.

What kinases are responsible for phosphorylating Histone H3 at Thr11?

Research has identified Dlk (death-associated protein like kinase) as a primary kinase responsible for phosphorylating Histone H3 at Thr11. In vitro studies demonstrated that Dlk specifically phosphorylates H3 at Thr11 rather than the more commonly phosphorylated Ser10 residue . Interestingly, the kinetics of Thr11 phosphorylation parallels that of the centromere-specific histone H3 variant CENP-A at Ser7, and evidence suggests Dlk may be capable of phosphorylating both sites, indicating a coordinated regulation mechanism for centromeric chromatin during mitosis . While other kinases like KimH3 have been shown to phosphorylate different sites on histone H3 (such as Ser10) during both interphase and mitosis , their role in Thr11 phosphorylation remains to be fully elucidated. This represents an area requiring further investigation to fully map the kinase network controlling this modification.

What are the recommended applications and dilutions for Phospho-Histone H3 (Thr11) Antibody?

Phospho-Histone H3 (Thr11) Antibody has been validated for multiple experimental applications with specific recommended dilutions to ensure optimal results. For Western blot (WB) analysis, the recommended dilution ranges from 1:500 to 1:1000, allowing for sensitive detection of the phosphorylated protein in cell and tissue lysates . For Enzyme-Linked Immunosorbent Assay (ELISA), a higher dilution of 1:2000 to 1:10000 is typically sufficient . The antibody is also suitable for immunofluorescence (IF) applications to visualize the spatial distribution of Thr11 phosphorylation, which is particularly valuable for investigating its centromeric localization during mitosis . Additionally, it has been validated for immunoprecipitation (IP) experiments to study protein interactions . When designing experiments, researchers should consider that this antibody recognizes human, mouse, and rat samples, allowing for comparative studies across these species .

How can I validate the specificity of Phospho-Histone H3 (Thr11) Antibody in my experiments?

Validating antibody specificity is crucial for ensuring reliable experimental results. For Phospho-Histone H3 (Thr11) Antibody, several approaches can be employed. First, peptide competition assays can be conducted where the antibody is pre-incubated with a competing phosphorylated peptide encompassing the Thr11 site (K-S-T(p)-G-G), which should block the signal in a concentration-dependent manner if the antibody is specific . Second, comparison with asynchronous versus mitotic cell populations can serve as a biological validation since Thr11 phosphorylation is predominantly a mitotic event; a strong enrichment should be observed in mitotic cells . Third, treatment with lambda phosphatase can be used to remove phosphate groups, which should eliminate antibody binding. Fourth, CRISPR-Cas9-mediated mutation of Thr11 to alanine in cell lines can serve as the ultimate specificity control. Finally, cross-reactivity with other phosphorylated residues, particularly Ser10, should be excluded by using appropriate controls and comparison with antibodies specific for other phosphorylation sites .

What is the optimal protocol for visualizing Phospho-Histone H3 (Thr11) localization using immunofluorescence?

For optimal visualization of Phospho-Histone H3 (Thr11) localization by immunofluorescence, the following detailed protocol is recommended:

• Cell preparation:

  • Grow cells on coverslips to 70-80% confluence

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Immunostaining:

  • Block with 5% BSA in PBS for 1 hour at room temperature

  • Incubate with Phospho-Histone H3 (Thr11) antibody at 1:200 dilution overnight at 4°C

  • Wash 3x with PBS (5 minutes each)

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

  • Counterstain DNA with DAPI or propidium iodide to visualize chromatin and determine mitotic stages

• Co-staining for centromere visualization:

  • For co-localization studies, include CREST serum (1:1000) during primary antibody incubation to visualize centromeres

  • Use distinct fluorophores for secondary antibodies to distinguish between H3-Thr11-P and centromere markers

• Image acquisition:

  • Use confocal microscopy for high-resolution imaging of centromeric dots

  • Capture z-stacks to ensure complete three-dimensional visualization of chromosomes

This protocol enables visualization of the characteristic centromeric localization pattern of Phospho-Histone H3 (Thr11) during mitosis .

How does Phospho-Histone H3 (Thr11) contribute to centromere function and mitotic progression?

The specific localization of Phospho-Histone H3 (Thr11) to centromeric regions during mitosis strongly suggests a specialized role in centromere function. Research indicates that this modification may contribute to several crucial aspects of mitotic progression. First, the timing of Thr11 phosphorylation (prophase to early anaphase) coincides with critical events in kinetochore assembly and chromosome segregation, suggesting it may participate in regulating these processes . Second, the co-localization with centromere markers revealed by confocal microscopy implies a potential role in organizing centromeric chromatin structure to facilitate kinetochore attachment . Third, the parallel kinetics with CENP-A phosphorylation at Ser7 suggests a coordinated regulatory mechanism for centromere function during mitosis .

Advanced research questions should explore how Thr11 phosphorylation affects centromere protein recruitment, microtubule attachment stability, and chromosome segregation fidelity. Moreover, the mutual exclusivity with Ser10 phosphorylation suggests a potential "histone code" at centromeres that warrants further investigation . Elucidating these mechanisms could provide insights into chromosomal instability in cancer and other diseases associated with mitotic defects.

How do the dynamics of Histone H3 Thr11 phosphorylation compare with other mitotic phosphorylation events?

The dynamics of Histone H3 Thr11 phosphorylation exhibit distinct patterns compared to other mitotic phosphorylation events, revealing a sophisticated regulatory network governing chromosome dynamics during cell division. Comparative analyses reveal that Thr11 phosphorylation occurs specifically from prophase to early anaphase, with the signal fading after anaphase onset . This differs from Ser10 phosphorylation, which appears earlier in late G2/early prophase and persists until anaphase .

A key distinguishing feature is the spatial distribution: Thr11 phosphorylation is predominantly localized to centromeric regions, while Ser10 phosphorylation is distributed throughout chromosomes, particularly in peripheral regions . This spatial specificity suggests differential functions, with Thr11 phosphorylation potentially playing a specialized role in centromere biology.

The relationship between different phosphorylation events appears complex. Evidence suggests that Ser10 and Thr11 phosphorylation may be mutually exclusive, indicating potential regulatory cross-talk . Furthermore, the kinetics of Thr11 phosphorylation parallels that of the centromere-specific histone H3 variant CENP-A at Ser7, suggesting coordinated regulation of centromeric chromatin during mitosis .

These observations point to a sophisticated "phosphorylation code" on histone H3 during mitosis, with different modifications serving distinct functions in chromosome condensation, centromere organization, and segregation.

What are common challenges when using Phospho-Histone H3 (Thr11) Antibody and how can they be overcome?

When working with Phospho-Histone H3 (Thr11) Antibody, researchers frequently encounter several challenges that require specific solutions:

• Weak signal detection: Since Thr11 phosphorylation is less abundant than Ser10 phosphorylation , signal detection can be challenging. To overcome this:

  • Optimize antibody concentration (start with 1:500 for WB and adjust as needed)

  • Enrich for mitotic cells using nocodazole or thymidine synchronization to increase target abundance

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blotting

  • Employ signal amplification systems for immunofluorescence

• Non-specific binding: To minimize background and ensure specificity:

  • Include competing phosphorylated peptide controls to confirm signal specificity

  • Optimize blocking conditions (try different blockers like 5% BSA or 5% milk)

  • Perform more stringent washing steps

  • Pre-adsorb antibody with non-phosphorylated peptide to remove antibodies that recognize unphosphorylated epitopes

• Phosphatase activity during sample preparation:

  • Add phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) to all buffers

  • Keep samples cold during preparation

  • Consider using phosphatase inhibitor cocktails specifically designed for histone research

• Cell cycle variability:

  • For consistent results, synchronize cells using double thymidine block or nocodazole treatment

  • Consider cell cycle analysis in parallel to correlate phosphorylation with specific phases

• Storage and stability issues:

  • Store antibody at -20°C in aliquots to avoid freeze-thaw cycles

  • Add 50% glycerol to storage buffer for long-term stability

These solutions should help researchers obtain reproducible and specific results when using Phospho-Histone H3 (Thr11) Antibody.

How can I quantify Phospho-Histone H3 (Thr11) levels in different experimental conditions?

Quantification of Phospho-Histone H3 (Thr11) levels across experimental conditions requires rigorous methodological approaches to ensure accuracy and reproducibility. Several complementary techniques can be employed:

• Western Blot Quantification:

  • Use recombinant phosphorylated standards to generate a calibration curve

  • Normalize Phospho-Histone H3 (Thr11) signal to total Histone H3 to account for loading variations

  • Employ digital imaging systems with wide dynamic range rather than film

  • Use densitometry software (ImageJ, ImageLab) with background subtraction

  • Perform statistical analysis across at least three biological replicates

• Flow Cytometry:

  • Fix and permeabilize cells appropriately to preserve phospho-epitopes

  • Co-stain with DNA dyes to correlate phosphorylation with cell cycle phase

  • Use mitotic markers (e.g., MPM-2) to specifically analyze mitotic populations

  • Calculate mean fluorescence intensity (MFI) or positive cell percentages

• Quantitative Immunofluorescence:

  • Capture images using identical acquisition parameters across samples

  • Measure fluorescence intensity at centromeres using specialized image analysis software

  • Count positive centromeres per cell in multiple fields

  • Normalize to CREST staining intensity when performing co-localization studies

• ELISA-based Methods:

  • Develop sandwich ELISA with capture antibody against H3 and detection antibody against Phospho-Thr11

  • Use purified phosphorylated peptide standards for quantification

  • Employ recommended dilutions (1:2000-1:10000) for optimal sensitivity

These approaches, used either individually or in combination, provide robust quantification of Phospho-Histone H3 (Thr11) levels across experimental conditions, enabling detailed analysis of this modification in various biological contexts.

What controls should be included when studying Phospho-Histone H3 (Thr11) in research experiments?

When studying Phospho-Histone H3 (Thr11), a comprehensive set of controls is essential to ensure experimental validity and interpretability. The following controls should be included:

• Positive Controls:

  • Mitotic cell populations enriched using nocodazole treatment, which show high levels of Thr11 phosphorylation

  • Recombinant Histone H3 phosphorylated at Thr11 by Dlk kinase in vitro

  • Cell lines known to express high levels of Thr11 phosphorylation (e.g., HeLa cells during mitosis)

• Negative Controls:

  • Asynchronous cell populations with low mitotic index, showing minimal Thr11 phosphorylation

  • Lambda phosphatase-treated samples to remove phosphorylation

  • Peptide competition assays using phosphorylated Thr11 peptide to block specific antibody binding

  • Primary antibody omission to assess secondary antibody non-specific binding

• Specificity Controls:

  • Parallel staining with antibodies against other phosphorylation sites (e.g., Ser10) to demonstrate site specificity

  • CRISPR-engineered cell lines with Thr11 to alanine mutation to confirm antibody specificity

  • Immunoprecipitation followed by mass spectrometry to confirm the modified residue

• Treatment Controls:

  • Kinase inhibitor controls (e.g., Dlk inhibitors) to demonstrate phosphorylation dependency

  • Cell cycle synchronization controls using different methods to rule out synchronization artifacts

  • Solvent controls for drug treatments

• Normalization Controls:

  • Total Histone H3 levels to normalize phosphorylation signals

  • Loading controls for Western blots (e.g., GAPDH, β-actin)

  • Internal reference standards for quantification

Implementing these controls ensures that observed changes in Phospho-Histone H3 (Thr11) levels are specific, reproducible, and biologically meaningful.

How does the function of Phospho-Histone H3 (Thr11) compare with Phospho-Histone H3 (Ser10) in cell cycle regulation?

The functional comparison between Phospho-Histone H3 (Thr11) and Phospho-Histone H3 (Ser10) reveals distinct roles in cell cycle regulation despite their proximity in the histone H3 N-terminal tail. These differences are summarized in the following comparative table:

These distinctions suggest a sophisticated regulatory system where different phosphorylation events on histone H3 serve complementary functions during cell division. While Ser10 phosphorylation appears to play a broader role in chromosome condensation throughout mitosis and gene activation during interphase, Thr11 phosphorylation likely serves a more specialized function in centromere biology during a specific window of mitosis . The mutual exclusivity of these modifications suggests potential cross-regulation mechanisms that warrant further investigation to fully understand the "histone phosphorylation code" governing cell cycle progression.

What are the implications of Phospho-Histone H3 (Thr11) research for understanding chromosomal instability in disease?

The study of Phospho-Histone H3 (Thr11) has significant implications for understanding chromosomal instability (CIN) in disease, particularly in cancer. Since Thr11 phosphorylation is concentrated at centromeres and occurs during specific stages of mitosis , disruption of this modification could compromise centromere function and chromosome segregation fidelity—processes directly linked to CIN.

Several mechanisms may connect aberrant Thr11 phosphorylation to disease pathogenesis:

• Centromere dysfunction: Improper Thr11 phosphorylation may disrupt centromere structure, affecting kinetochore assembly and microtubule attachment, leading to chromosome missegregation . This can result in aneuploidy, a common feature of many cancers.

• Mitotic checkpoint abnormalities: The specific timing of Thr11 phosphorylation suggests potential involvement in mitotic checkpoint regulation . Dysregulation could allow cells with improper chromosomal attachments to progress through mitosis, promoting genomic instability.

• Altered epigenetic landscapes: As a histone modification, aberrant Thr11 phosphorylation may disrupt the epigenetic landscape at centromeres, affecting centromere identity and function over multiple cell divisions.

• Kinase dysregulation: Abnormal activity of Dlk or other kinases responsible for Thr11 phosphorylation could contribute to disease progression . Similar to how KimH3 dysregulation correlates with cancer patient survival , alterations in Thr11 kinases might have prognostic significance.

Future research should explore Thr11 phosphorylation patterns in patient samples from diseases characterized by CIN, develop diagnostic tools based on this modification, and investigate therapeutic approaches targeting the responsible kinases or downstream pathways to potentially mitigate chromosomal instability in disease contexts.

How can advanced techniques like ChIP-seq and phosphoproteomics enhance our understanding of Phospho-Histone H3 (Thr11) biology?

Advanced molecular techniques are revolutionizing our understanding of histone modifications, including Phospho-Histone H3 (Thr11). These methodologies offer unprecedented insights into the genomic distribution, regulatory mechanisms, and functional implications of this modification:

• ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing):

  • Maps the precise genomic locations of Thr11 phosphorylation with base-pair resolution

  • Reveals whether Thr11 phosphorylation occupies specific centromeric sequences or extends to pericentromeric regions

  • Enables correlation with other histone marks, DNA sequences, and chromatin-associated proteins

  • Can be combined with cell synchronization to track dynamic changes throughout mitosis

  • Allows comparison between normal and disease states to identify pathological alterations

• Phosphoproteomics:

  • Quantifies global levels of Thr11 phosphorylation in different cellular contexts

  • Identifies novel proteins that interact specifically with phosphorylated Thr11

  • Characterizes phosphorylation dynamics during different cell cycle phases

  • Maps kinase-substrate relationships to identify all kinases capable of phosphorylating H3 at Thr11

  • Enables discovery of phosphatases responsible for removing this modification

• Integrative Multi-omics Approaches:

  • Combining ChIP-seq, phosphoproteomics, transcriptomics, and proteomics to create comprehensive models

  • Correlating Thr11 phosphorylation patterns with transcriptional outputs and cellular phenotypes

  • Network analysis to position Thr11 phosphorylation within broader regulatory circuits

• Advanced Microscopy Techniques:

  • Super-resolution microscopy to visualize nanoscale distribution at centromeres

  • Live-cell imaging with phospho-specific fluorescent probes to track dynamics in real-time

  • FRET (Förster Resonance Energy Transfer) to study interactions between modified histones and other proteins

These technologies, individually and in combination, promise to transform our understanding of Phospho-Histone H3 (Thr11) from a static marker to a dynamic component of chromatin regulation with precise spatiotemporal characteristics and functional implications in normal biology and disease.

What are the most promising future directions in Phospho-Histone H3 (Thr11) research?

The field of Phospho-Histone H3 (Thr11) research stands at an exciting frontier with several promising directions for future investigation. First, the centromere-specific localization of Thr11 phosphorylation warrants deeper exploration of its role in kinetochore assembly and chromosome segregation mechanisms . Targeted mutations of Thr11 in model organisms could reveal phenotypic consequences and elucidate its essential functions. Second, given the association between histone phosphorylation dysregulation and cancer , comprehensive profiling of Thr11 phosphorylation across cancer types could identify potential biomarkers and therapeutic targets. Third, identifying the complete set of kinases and phosphatases regulating Thr11 phosphorylation beyond Dlk would provide a more comprehensive understanding of its regulation .

The mutual exclusivity with Ser10 phosphorylation suggests a complex "phosphorylation code" that merits further investigation to understand how different histone modifications are coordinated during mitosis . Additionally, potential interplay between Thr11 phosphorylation and other histone modifications such as methylation or acetylation remains largely unexplored. Finally, the development of small molecule inhibitors targeting kinases responsible for Thr11 phosphorylation, similar to those developed for other histone kinases , could provide valuable research tools and potential therapeutic approaches for diseases characterized by chromosomal instability.

By pursuing these research directions, scientists can advance our understanding of fundamental chromatin biology while potentially uncovering new approaches to diagnose and treat diseases associated with mitotic dysfunction.

How can researchers optimize their experimental design when studying Phospho-Histone H3 (Thr11)?

To optimize experimental design when studying Phospho-Histone H3 (Thr11), researchers should implement a comprehensive strategy that accounts for the unique characteristics of this modification:

• Cell Synchronization Approaches:

  • Employ multiple synchronization methods (double thymidine block, nocodazole, CDK1 inhibitors) to rule out method-specific artifacts

  • Include time-course analyses to capture the dynamic window of Thr11 phosphorylation (prophase to early anaphase)

  • Consider single-cell approaches to account for cell-to-cell variability in mitotic progression

• Antibody Validation Strategy:

  • Perform rigorous specificity testing using peptide competition assays with both phosphorylated and non-phosphorylated peptides

  • Include phosphatase treatments as negative controls

  • Compare results using antibodies from different sources where possible

  • Consider generating conditional knockin cell lines expressing H3 with Thr11 to alanine mutations as ultimate specificity controls

• Multidisciplinary Technical Approach:

  • Combine immunofluorescence for spatial information with Western blotting for quantification

  • Implement super-resolution microscopy to precisely analyze centromeric localization

  • Use live-cell imaging when possible to capture real-time dynamics

  • Consider ChIP-seq to map genomic distribution at high resolution

• Appropriate Comparisons and Controls:

  • Always include both Phospho-Histone H3 (Ser10) and total Histone H3 analyses for comparison

  • Use CREST serum co-staining to definitively identify centromeric regions

  • Include kinase inhibitor controls to establish causal relationships

• Statistical Considerations:

  • Perform power calculations to determine appropriate sample sizes

  • Analyze sufficient numbers of cells (typically >100 per condition)

  • Use appropriate statistical tests for data analysis

  • Implement blinded quantification to prevent observer bias

By incorporating these elements into experimental design, researchers can generate robust and reproducible data on Phospho-Histone H3 (Thr11) that advances our understanding of its biological functions and potential clinical applications.

What collaborative approaches might accelerate progress in understanding the role of Phospho-Histone H3 (Thr11) in cellular processes?

Accelerating progress in understanding Phospho-Histone H3 (Thr11) biology requires innovative collaborative approaches that transcend traditional research boundaries. Several strategic collaborations could particularly advance the field:

• Interdisciplinary Research Consortia:

  • Combining expertise from chromosome biologists, structural biologists, and computational scientists

  • Integrating cell biologists studying mitosis with cancer researchers examining chromosomal instability

  • Establishing shared resources for specialized reagents and model systems

  • Creating standardized protocols to enable cross-laboratory validation

• Technology-Driven Partnerships:

  • Collaborations between antibody developers and academic researchers to create higher-specificity reagents

  • Partnerships with proteomics facilities to identify novel Thr11-interacting proteins

  • Engagement with advanced microscopy centers to implement cutting-edge imaging approaches

  • Collaborations with structural biology groups to determine atomic-level changes induced by phosphorylation

• Clinical-Basic Science Bridges:

  • Biobanking initiatives to collect and characterize patient samples for Thr11 phosphorylation patterns

  • Translational research programs linking basic Thr11 findings with clinical observations

  • Collaborations testing kinase inhibitors in patient-derived organoids or xenografts

  • Registry studies correlating Thr11 phosphorylation with clinical outcomes

• Open Science Initiatives:

  • Development of public databases documenting Thr11 phosphorylation across cell types and conditions

  • Pre-registered studies to enhance reproducibility of key findings

  • Resource sharing through repositories of validated reagents, cell lines, and protocols

  • Crowd-sourced analysis of complex datasets (e.g., phosphorylation patterns in cancer samples)

• Educational Collaborations:

  • Workshop series to disseminate specialized techniques for studying histone phosphorylation

  • Cross-training opportunities between laboratories with complementary expertise

  • Collaborative grant mechanisms requiring multi-institutional approaches