Phospho-HIST1H3A (S10) Antibody

What is a Phospho-Histone H3 (S10) antibody and what does it detect?

Phospho-Histone H3 (S10) antibodies are specifically designed to recognize histone H3 protein only when phosphorylated at the serine 10 position. This post-translational modification occurs predominantly during mitosis and serves as a critical marker for chromosome condensation and cell division. These antibodies can be produced in various host animals, with rabbit and mouse being the most common, and are available in both polyclonal and monoclonal forms . The molecular weight of the detected protein is approximately 17 kDa for human histone H3 .

What are the common laboratory applications for Phospho-Histone H3 (S10) antibodies?

These antibodies have versatility across multiple experimental platforms including:

• Western blotting (WB) for protein detection and quantification

• Immunohistochemistry (IHC) for tissue section analysis

• Immunofluorescence (IF) for cellular localization studies

• Enzyme-linked immunosorbent assay (ELISA) for quantitative measurements

• Chromatin immunoprecipitation (ChIP) for analyzing protein-DNA interactions

• Flow cytometry for cell cycle analysis and mitotic index determination

• Dot blot for rapid protein detection

• Multiplex assays for simultaneous detection of multiple targets

What is the significance of Histone H3 phosphorylation at Serine 10 in biological systems?

Histone H3 phosphorylation at Serine 10 serves as a critical regulatory mechanism in chromatin dynamics. During mitosis, this modification facilitates chromosome condensation essential for proper cell division. Additionally, this phosphorylation plays roles in transcriptional activation and is implicated in cellular responses to various stimuli. The modification is dynamically regulated, increasing dramatically during the G2/M transition and decreasing upon exit from mitosis, making it an excellent marker for mitotic cells in research and potentially in diagnostic applications .

How should researchers optimize antibody dilutions for different experimental applications?

Optimal dilution ranges vary significantly based on the application:

These ranges provide starting points; researchers should perform titration experiments to determine optimal concentrations for their specific experimental conditions .

What controls should be included when using Phospho-Histone H3 (S10) antibodies?

Rigorous experimental design requires appropriate controls:

• Positive controls: Colcemid-treated or mitotic HeLa cells show high levels of H3S10 phosphorylation

• Negative controls: Non-mitotic cells or samples treated with phosphatase

• Isotype controls: Matching IgG from the same species as the primary antibody

• Blocking peptide controls: Peptides containing phosphorylated S10 should block specific binding

• Cross-reactivity controls: Testing against similar modifications (e.g., phospho-S28, phospho-T11) to confirm specificity

Peptide inhibition analysis has demonstrated that detection of histone H3 can be diminished by histone H3 peptides containing phospho-serine 10, but not by peptides containing phospho-serine 28 or unmodified histone H3 sequences .

What are the recommended storage and handling conditions for maintaining antibody activity?

To preserve antibody functionality:

• Long-term storage: -20°C for up to one year

• Short-term/frequent use: 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they degrade antibody quality

• Most preparations contain glycerol (30-50%) to prevent freeze damage

• Many commercial preparations include stabilizers such as BSA (0.5-1%) and preservatives like sodium azide (0.02%)

• Working dilutions should be prepared fresh and used within 24 hours for optimal results

How can Phospho-Histone H3 (S10) antibodies be utilized in ChIP assays to study chromatin dynamics?

Chromatin immunoprecipitation (ChIP) with Phospho-Histone H3 (S10) antibodies provides valuable insights into the genomic distribution of this modification:

ChIP protocols typically require:

• 1-10 μg of antibody per ChIP experiment

• Sheared chromatin from approximately 10,000 cells

• IgG controls at equivalent concentrations

• Quantitative PCR analysis with primers for genomic regions of interest

Research has shown that recovery of immunoprecipitated DNA (expressed as percentage of input) varies between genes and is significantly increased in colcemid-treated (mitosis-arrested) cells compared to untreated cells. For example, ChIP assays performed with primers for promoters of active genes like c-fos and RPL30, as well as the Sat2 satellite repeat region, show differential enrichment patterns that reflect the biological distribution of H3S10 phosphorylation .

What considerations are important when selecting between monoclonal and polyclonal Phospho-Histone H3 (S10) antibodies?

The choice between monoclonal and polyclonal antibodies depends on research objectives:

Monoclonal Advantages:

• Greater specificity and batch-to-batch consistency

• Lower background in applications like immunofluorescence

• Precise epitope recognition (e.g., clone 3H10 specifically recognizes H3S10ph)

Polyclonal Advantages:

• Recognition of multiple epitopes improves signal strength

• Greater tolerance to minor protein denaturation

• Often perform better in applications like western blotting

• May detect the target across more species due to broader reactivity

For highly specific applications requiring reproducibility across multiple experiments, monoclonal antibodies may be preferable, while polyclonals might offer superior signal detection in applications where sensitivity is paramount.

How can Phospho-Histone H3 (S10) antibodies be implemented in multiplex assays?

Multiplex approaches allow simultaneous detection of multiple targets:

• Beadlyte Histone-Peptide Specificity Assays use Luminex microspheres conjugated to different histone peptides for multiplexed detection

• Co-staining experiments can combine Phospho-Histone H3 (S10) antibodies with other cell cycle markers (e.g., Ki-67, PCNA)

• Species-specific secondary antibodies with different fluorophores enable multiple target visualization

• Flow cytometry multiplex protocols can simultaneously assess cell cycle stage, DNA content, and H3S10 phosphorylation

Optimization is critical for multiplex applications to minimize cross-reactivity and ensure signal specificity. Proper titration of antibodies and sequential staining protocols may be necessary depending on the experimental design.

What are common issues encountered when using Phospho-Histone H3 (S10) antibodies and how can they be resolved?

Researchers frequently encounter several challenges:

For Western blot applications specifically, extraction methods that preserve phosphorylation status (like acid extraction for histones) are critical for successful detection .

How can researchers validate the specificity of Phospho-Histone H3 (S10) antibodies?

Rigorous validation ensures reliable results:

• Peptide competition assays with phosphorylated and non-phosphorylated peptides

• Comparison of staining patterns in mitotic versus interphase cells

• Testing reactivity against various histone modifications to ensure specificity

• Dot blot analysis with peptides containing different modifications

• ELISA-based quantification against specific target peptides

• Beadlyte histone-peptide specificity assays with various modifications

For example, one validation showed that dilutions ranging from 1:1,000 to 1:81,000 of the antibody specifically detected only peptides containing phospho-serine 10 when tested against various histone H3 peptides .

What considerations are important when quantifying Phospho-Histone H3 (S10) signals in imaging applications?

Accurate quantification requires:

• Consistent exposure settings across all samples and controls

• Proper background subtraction specific to each image

• Identification of true positives (typically showing distinct nuclear/chromosomal patterns)

• Normalization to appropriate cellular markers or total cell counts

• Software-based intensity measurements with defined thresholds

• Statistical analysis accounting for cell cycle distribution in populations

Phospho-Histone H3 (S10) typically shows intense staining of condensed chromosomes in mitotic cells, while interphase cells should show minimal or no signal, providing an internal control within mixed populations .

How are Phospho-Histone H3 (S10) antibodies utilized in cancer research and diagnostics?

These antibodies serve important roles in oncology research:

• Assessment of mitotic index in tumor samples, correlating with aggressiveness

• Evaluation of anti-mitotic drug efficacy in cancer cell lines

• Identification of aberrant cell cycle regulation in malignant tissues

• Potential diagnostic marker for distinguishing tumor grades

• Studying the effects of epigenetic modifiers on cancer cell proliferation

The specific and robust detection of mitotic cells using these antibodies allows for standardized quantification of proliferation rates across different cancer types and treatment conditions .

What recent methodological advances have improved Phospho-Histone H3 (S10) antibody applications?

Technical innovations continue to enhance research capabilities:

• Flow cytometry protocols allowing simultaneous DNA content and H3S10ph analysis

• Improved chromatin preparation methods for ChIP sequencing applications

• ZooMAb® technology providing enhanced reproducibility for rabbit monoclonal antibodies

• Combined detection of multiple histone modifications (e.g., H3K9ac/H3S10ph) to study cross-talk

• Optimized fixation protocols preserving phospho-epitopes in tissue specimens

How does the choice of fixation and sample preparation affect Phospho-Histone H3 (S10) antibody performance?

Sample preparation critically influences results:

• Ethanol/acetic acid fixation (95% ethanol, 5% acetic acid) preserves phospho-epitopes effectively

• Formalin fixation may mask epitopes, requiring antigen retrieval methods

• Permeabilization with 0.1% Triton X-100 improves antibody accessibility

• Rapid fixation is essential as phosphorylation status can change quickly

• Phosphatase inhibitors should be included in all extraction buffers

• Different antibody clones may perform optimally with specific fixation methods

In immunocytochemistry applications, a concentration of 0.2 μg/mL has been shown to effectively detect positive chromosome immunostaining in mitotic A431 and HeLa cells when fixed with 95% ethanol and 5% acetic acid and permeabilized with 0.1% Triton X-100 .