Phospho-HIST1H1B (S172) Antibody

What is HIST1H1B and why is its phosphorylation at S172 significant?

HIST1H1B, also known as Histone H1.5 (Histone H1a/H1b/H1s-3), is a linker histone that binds to DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. It plays a crucial role in the condensation of nucleosome chains into higher-order structured fibers and functions as a regulator of gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation .

Phosphorylation at serine 172 (S172) is particularly significant as this post-translational modification alters the protein's interaction with DNA and other nuclear proteins. This specific phosphorylation event has been implicated in chromatin dynamics during cell cycle progression, DNA damage response, and transcriptional regulation. The phosphorylation status at S172 can significantly impact gene expression patterns and cellular processes, making it an important marker for epigenetic regulation studies .

What experimental applications is the Phospho-HIST1H1B (S172) Antibody validated for?

Based on current validation data, the Phospho-HIST1H1B (S172) Antibody has been specifically validated for the following applications:

The antibody has been rigorously tested for specificity and sensitivity in these applications, with particular effectiveness in detecting phosphorylated HIST1H1B in human samples . While some cross-reactivity with mouse and rat samples may be possible due to sequence conservation, additional validation is recommended when working with non-human species.

How does phosphorylation at S172 differ from other phosphorylation sites on HIST1H1B?

HIST1H1B contains multiple phosphorylation sites, including T10, T154, and S172, each with distinct functions and regulation patterns:

S172 phosphorylation is particularly distinctive as it often occurs in response to cellular stressors and DNA damage, whereas T10 phosphorylation is more strictly associated with mitotic progression. Understanding these different phosphorylation patterns is crucial for interpreting experimental results and designing targeted studies of histone regulation .

What are the optimal conditions for Western blot detection of Phospho-HIST1H1B (S172)?

For optimal Western blot detection of Phospho-HIST1H1B (S172), the following protocol is recommended:

• Sample preparation:

  • Extract histones using an acid extraction method (0.2N HCl or 0.4N H2SO4)

  • Include phosphatase inhibitors (10mM sodium fluoride, 1mM sodium orthovanadate, 10mM β-glycerophosphate) in all buffers

  • Use freshly prepared samples whenever possible to prevent dephosphorylation

• Gel electrophoresis:

  • Use 15% SDS-PAGE gels to achieve good separation of histone proteins

  • Load 10-20μg of histone extract per lane

  • Include positive controls (cells treated with phosphatase inhibitors) and negative controls (samples treated with lambda phosphatase)

• Transfer and detection:

  • Transfer to PVDF membrane (0.2μm pore size) at 30V overnight at 4°C

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with Phospho-HIST1H1B (S172) antibody at 1:1000 dilution in 5% BSA/TBST overnight at 4°C

  • Use an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

  • Develop using enhanced chemiluminescence (ECL) substrates

• Expected results:

  • Phospho-HIST1H1B (S172) appears at approximately 23-25 kDa

  • Signal intensity may vary based on cell cycle stage and treatment conditions

What controls should be included when working with Phospho-HIST1H1B (S172) Antibody?

To ensure experimental rigor and valid interpretation of results, the following controls should be included:

How can I optimize immunocytochemistry (ICC) experiments with this antibody?

For successful ICC experiments using Phospho-HIST1H1B (S172) Antibody, consider the following optimization steps:

• Fixation and permeabilization:

  • Use 4% paraformaldehyde for 15 minutes at room temperature

  • Avoid methanol fixation as it can extract phosphorylated proteins

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

• Blocking and antibody incubation:

  • Block with 5% normal goat serum and 1% BSA in PBS for 1 hour

  • Use Phospho-HIST1H1B (S172) antibody at 1:50 dilution for initial testing

  • Incubate overnight at 4°C in a humidified chamber

  • Include phosphatase inhibitors (10mM NaF, 1mM Na3VO4) in all solutions

• Signal detection and visualization:

  • Use fluorescent secondary antibodies (Alexa Fluor conjugates recommended)

  • Counterstain nuclei with DAPI

  • Examine at different exposure settings to capture optimal signal

• Critical considerations:

  • Avoid phosphate buffers during fixation as they can interfere with phospho-epitopes

  • Process control and experimental samples simultaneously under identical conditions

  • Include peptide competition controls to verify signal specificity

  • Co-stain with markers of cellular compartments or cell cycle phases for contextual analysis

A recommended dilution series (1:10, 1:25, 1:50, 1:100) should be tested to determine optimal antibody concentration for your specific cell type and experimental conditions.

How do I quantify changes in HIST1H1B S172 phosphorylation levels?

Quantifying changes in HIST1H1B S172 phosphorylation requires careful normalization and statistical analysis:

How do cell cycle phases affect HIST1H1B S172 phosphorylation patterns?

HIST1H1B S172 phosphorylation exhibits distinct patterns across cell cycle phases:

These dynamics make it essential to consider cell cycle synchronization or co-staining with cell cycle markers (e.g., cyclin B1, phospho-histone H3) when interpreting experimental results. Changes in phosphorylation levels should be analyzed in the context of potential cell cycle alterations induced by experimental treatments .

How can I distinguish between specific S172 phosphorylation signal and antibody cross-reactivity?

Ensuring signal specificity requires multiple validation approaches:

• Peptide competition assays:

  • Pre-incubate the antibody with phosphorylated S172 peptide (10-100 μg/ml)

  • In parallel, pre-incubate with non-phosphorylated S172 peptide

  • A specific signal should be blocked by phospho-peptide but not by non-phospho-peptide

• Phosphatase treatment controls:

  • Treat duplicate samples with lambda phosphatase

  • Specific phospho-signals should be eliminated after phosphatase treatment

• Genetic approaches:

  • Use CRISPR/Cas9 to generate S172A mutants (prevents phosphorylation)

  • Compare wild-type and mutant signals in identical experimental conditions

• Cross-reactivity testing:

  • Test antibody against recombinant histones with different phosphorylation states

  • Examine reactivity with S172-analogous sites in other H1 variants

  • Consider dot blot arrays with various phosphorylated and non-phosphorylated peptides

• Mass spectrometry validation:

  • For definitive confirmation, immunoprecipitate using the phospho-antibody

  • Analyze by mass spectrometry to confirm the presence of the S172 phosphopeptide

What signaling pathways regulate HIST1H1B S172 phosphorylation?

HIST1H1B S172 phosphorylation is regulated by multiple signaling pathways:

Understanding these regulatory pathways helps design experiments to modulate HIST1H1B S172 phosphorylation. For example, treating cells with ATM/ATR inhibitors (e.g., KU-55933) can block DNA damage-induced phosphorylation, while PKA activators (e.g., forskolin) can enhance basal phosphorylation levels .

How does HIST1H1B S172 phosphorylation interact with other epigenetic modifications?

HIST1H1B S172 phosphorylation operates within a complex network of epigenetic modifications:

• Interactions with other histone modifications:

  • S172 phosphorylation often correlates positively with H3S10 phosphorylation

  • Negatively correlates with H3K9 trimethylation in heterochromatic regions

  • Can influence H2AX phosphorylation during DNA damage response

• Impact on chromatin remodeling:

  • Phosphorylated HIST1H1B shows reduced affinity for DNA

  • Facilitates binding of chromatin remodeling complexes (SWI/SNF, ISWI)

  • Creates permissive chromatin state for transcription factor binding

• Relationship with DNA methylation:

  • S172 phosphorylation can disrupt HIST1H1B interaction with DNA methyltransferases

  • May affect maintenance of DNA methylation patterns during replication

  • Altered S172 phosphorylation correlates with DNA methylation changes in cancer cells

• Cross-talk with non-histone proteins:

  • Phosphorylated S172 creates binding sites for 14-3-3 proteins

  • Modulates interaction with HP1 proteins and heterochromatin formation

  • Can affect recruitment of DNA repair machinery after damage

These interactions highlight the importance of studying S172 phosphorylation in the broader context of the epigenetic landscape, using approaches like sequential ChIP (ChIP-reChIP) or mass spectrometry-based proteomics to analyze modification co-occurrence .

What is the role of HIST1H1B S172 phosphorylation in disease pathology?

HIST1H1B S172 phosphorylation has been implicated in several disease contexts:

• Cancer:

  • Elevated S172 phosphorylation observed in aggressive breast and prostate cancers

  • Correlates with increased proliferation and poor prognosis

  • May contribute to genomic instability through altered chromatin structure

  • Potential biomarker for response to certain chemotherapeutic agents

• Neurodegenerative disorders:

  • Altered HIST1H1B phosphorylation patterns in Alzheimer's disease brain samples

  • Possible link to dysregulated gene expression in neuronal cells

  • May contribute to aberrant protein aggregation through chromatin structure changes

• Inflammatory diseases:

  • Dynamic changes in S172 phosphorylation during inflammatory responses

  • Potential role in regulating cytokine gene expression

  • Associated with altered immune cell function and differentiation

• Developmental disorders:

  • Critical for proper embryonic development and cellular differentiation

  • Disrupted phosphorylation patterns linked to developmental abnormalities

  • Important for establishing and maintaining cell type-specific gene expression patterns

These disease associations make HIST1H1B S172 phosphorylation a potential therapeutic target. Compounds that modulate this phosphorylation event, either directly or through upstream kinases, are being investigated for their clinical potential. Chromatin-modifying drugs like histone deacetylase inhibitors have been shown to alter HIST1H1B phosphorylation patterns and may derive part of their efficacy through this mechanism .

What are common challenges when working with Phospho-HIST1H1B (S172) Antibody and how can they be addressed?

Researchers frequently encounter several challenges when working with phospho-specific antibodies like Phospho-HIST1H1B (S172):

• Low signal intensity:

  • Cause: Rapid dephosphorylation during sample preparation, insufficient antibody concentration

  • Solution: Include multiple phosphatase inhibitors in all buffers, increase antibody concentration or incubation time, use signal enhancement systems

• High background:

  • Cause: Non-specific binding, excessive antibody concentration, inadequate blocking

  • Solution: Optimize blocking conditions (test different blocking agents like BSA vs. casein), reduce antibody concentration, increase washing stringency (add 0.1% SDS or 0.5M NaCl to wash buffer)

• Inconsistent results between experiments:

  • Cause: Variable phosphorylation status due to cell culture conditions, passage number, or cell cycle distribution

  • Solution: Standardize culture conditions, use synchronized cells, include multiple biological replicates, document cell confluency and passage number

• Cross-reactivity with other phosphorylated histones:

  • Cause: Sequence similarity between different histone variants

  • Solution: Validate with peptide competition assays, include appropriate knockout/knockdown controls, confirm results with alternative detection methods

• Poor reproducibility in fixed tissue samples:

  • Cause: Phospho-epitope destruction during fixation or processing

  • Solution: Minimize fixation time, use phospho-friendly fixatives (avoid prolonged formalin fixation), consider antigen retrieval methods optimized for phospho-epitopes

How can I optimize ChIP experiments using Phospho-HIST1H1B (S172) Antibody?

Chromatin immunoprecipitation (ChIP) with Phospho-HIST1H1B (S172) requires specific optimization:

• Crosslinking and chromatin preparation:

  • Use dual crosslinking (1% formaldehyde followed by ethylene glycol bis-succinimidylsuccinate)

  • Include phosphatase inhibitors in all buffers

  • Sonicate to achieve chromatin fragments of 200-500bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Pre-clear chromatin with protein A/G beads

  • Use 3-5μg antibody per ChIP reaction

  • Incubate overnight at 4°C with rotation

  • Include IgG negative control and total H3 positive control IPs

  • Consider sequential ChIP for co-occurrence with other modifications

• Washing and elution:

  • Use stringent wash conditions (increase salt concentration gradually)

  • Elute at 65°C to maintain phospho-epitope integrity

  • Reverse crosslinks overnight at 65°C

• Analysis recommendations:

  • Use qPCR for candidate regions and ChIP-seq for genome-wide analysis

  • Compare enrichment patterns with other histone modifications

  • Integrate with transcriptomic data to correlate with gene expression

  • Analyze distribution at promoters, enhancers, and gene bodies separately

• Expected patterns:

  • Phospho-HIST1H1B (S172) typically enriches at transcriptionally active regions

  • Shows cell cycle-dependent occupancy patterns

  • Often correlates with RNA Polymerase II occupancy

  • May show differential patterns at housekeeping vs. inducible genes

How do I interpret contradictory results between different detection methods?

When facing contradictory results between different detection methods for Phospho-HIST1H1B (S172):

• Understand method-specific limitations:

  • Western blot: Measures bulk levels, may miss cell-specific or locus-specific changes

  • Immunofluorescence: Provides spatial information but is semi-quantitative

  • ChIP: Reveals genomic localization but can be affected by antibody specificity

  • ELISA: Highly quantitative but lacks spatial or genomic information

  • Mass spectrometry: Most specific but requires specialized equipment and expertise

• Systematic troubleshooting approach:

  • Verify antibody specificity in each method independently

  • Test different sample preparation methods to preserve phosphorylation

  • Consider fixation artifacts in immunofluorescence

  • Evaluate effectiveness of extraction methods for different cellular compartments

• Biological interpretation of discrepancies:

  • Different subpopulations of HIST1H1B may show varying phosphorylation

  • Consider dynamic turnover rates and temporal factors

  • Evaluate whether contradictions reflect method sensitivity or biological reality

  • Spatial reorganization may explain differences between bulk and localized measurements

• Resolution strategies:

  • Use orthogonal approaches (e.g., genetic manipulation of S172 site)

  • Employ kinase inhibitors to modulate phosphorylation

  • Consider combined approaches (e.g., Fluorescence-Activated Cell Sorting followed by Western blot)

  • Develop internal standards for cross-method calibration

When reporting contradictory results, transparently document the experimental conditions for each method and discuss possible explanations for the observed differences.

What emerging technologies can enhance the study of HIST1H1B S172 phosphorylation?

Several cutting-edge technologies are expanding our ability to study HIST1H1B S172 phosphorylation:

• Single-cell epigenomics:

  • Single-cell CUT&Tag/CUT&RUN for genome-wide mapping of S172 phosphorylation

  • Correlation with transcriptional heterogeneity at single-cell resolution

  • Reveals cell state transitions and rare subpopulations

• Live-cell imaging approaches:

  • FRET-based biosensors for real-time monitoring of S172 phosphorylation

  • Optogenetic tools to induce targeted phosphorylation/dephosphorylation

  • Super-resolution microscopy to visualize chromatin reorganization

• Proximity labeling methods:

  • BioID or APEX2 fused to HIST1H1B to identify interactors specific to phosphorylated state

  • ChIP-SICAP to identify proteins co-occupying genomic loci with phosphorylated HIST1H1B

  • Reveals dynamic protein complexes regulated by phosphorylation

• CRISPR-based epigenome editing:

  • Targeted recruitment of kinases/phosphatases to specific genomic loci

  • Creation of designer phosphorylation patterns to assess functional consequences

  • High-throughput screening of phosphorylation effects on gene expression

• Structural biology approaches:

  • Cryo-EM of nucleosomes containing phosphorylated HIST1H1B

  • Molecular dynamics simulations to predict structural changes

  • Reveals mechanism of phosphorylation-induced chromatin remodeling

These technologies promise to advance our understanding of the spatiotemporal dynamics and functional consequences of HIST1H1B S172 phosphorylation in normal and disease states.

How can I design experiments to study the functional impact of S172 phosphorylation?

To elucidate the functional significance of HIST1H1B S172 phosphorylation, consider these experimental approaches:

• Genetic manipulation approaches:

  • Generate CRISPR knock-in cell lines with S172A (phospho-null) or S172E/D (phospho-mimetic) mutations

  • Create inducible expression systems for wild-type vs. mutant HIST1H1B

  • Perform rescue experiments in HIST1H1B knockdown backgrounds

• Functional readouts to assess:

  • Transcriptional changes (RNA-seq, nascent RNA-seq)

  • Chromatin accessibility (ATAC-seq, DNase-seq)

  • Replication timing and efficiency (Repli-seq, EdU incorporation)

  • DNA damage response (γH2AX foci, comet assays)

  • Cell cycle progression (flow cytometry, live-cell imaging)

• Context-dependent experiments:

  • Compare phosphorylation dynamics during normal vs. stressed conditions

  • Assess cell type-specific functions in differentiated vs. stem cells

  • Evaluate impact during development or cellular reprogramming

  • Study consequences during disease progression models

• Integrative approaches:

  • Correlate phosphorylation status with multi-omics data (transcriptome, proteome, metabolome)

  • Use network analysis to identify pathways affected by S172 phosphorylation

  • Develop predictive models for phosphorylation-dependent gene regulation

• Translational experiments:

  • Assess correlation between S172 phosphorylation and clinical outcomes

  • Test phosphorylation-modulating compounds in disease models

  • Evaluate potential as biomarker for disease progression or treatment response

These experimental strategies, when combined with appropriate controls and quantitative analysis, can provide comprehensive insights into the functional significance of HIST1H1B S172 phosphorylation .

What are the current gaps in our understanding of HIST1H1B S172 phosphorylation?

Despite significant advances, several knowledge gaps remain in our understanding of HIST1H1B S172 phosphorylation:

• Regulatory mechanisms:

  • Identity of all kinases and phosphatases that directly modify S172

  • Signaling pathways that connect cellular stimuli to S172 phosphorylation

  • Temporal dynamics of phosphorylation/dephosphorylation cycles

• Functional consequences:

  • Gene-specific effects of S172 phosphorylation

  • Impact on higher-order chromatin structure beyond nucleosome level

  • Role in cell type-specific gene expression programs

  • Contribution to cellular memory and epigenetic inheritance

• Disease relevance:

  • Causal relationship between altered S172 phosphorylation and disease progression

  • Potential as therapeutic target or biomarker

  • Tissue-specific consequences of dysregulated phosphorylation

• Technical limitations:

  • Need for improved antibody specificity and sensitivity

  • Challenges in detecting dynamic changes at single-cell resolution

  • Difficulty distinguishing functional consequences from correlation

Addressing these gaps will require interdisciplinary approaches combining biochemistry, genomics, structural biology, and computational modeling.

What best practices should researchers follow when working with phospho-specific histone antibodies?

To ensure robust and reproducible research with phospho-specific histone antibodies:

• Antibody validation:

  • Always validate antibody specificity with peptide competition assays

  • Test against phosphatase-treated samples

  • Include phospho-null mutants as negative controls when possible

  • Document lot-to-lot variation and validate each new lot

• Experimental design:

  • Include biological replicates (minimum n=3)

  • Incorporate appropriate positive and negative controls

  • Use multiple detection methods to confirm key findings

  • Consider cell cycle effects in experimental design and analysis

• Sample handling:

  • Use fresh samples whenever possible

  • Include phosphatase inhibitors in all buffers

  • Standardize sample collection and processing protocols

  • Document and control for variables that might affect phosphorylation status

• Data reporting:

  • Provide detailed methods including antibody catalog numbers, dilutions, and incubation conditions

  • Share original, unprocessed images

  • Describe quantification methods and statistical analyses in detail

  • Report negative and contradictory results

  • Consider data deposition in public repositories

• Interpretation:

  • Distinguish correlation from causation

  • Consider alternative explanations for observed phenotypes

  • Contextualize findings within broader epigenetic regulation

  • Acknowledge limitations and technical constraints