Phospho-HIST1H2BC (S14) Antibody

What is the biological function of H2B S14 phosphorylation in chromatin regulation?

Histone H2B serine 14 phosphorylation (H2BS14p) serves as a key regulatory modification within the nucleosome structure. As a core component of nucleosomes, histones wrap and compact DNA into chromatin, thereby limiting DNA accessibility to cellular machineries that require DNA as a template . The phosphorylation of H2B at S14 specifically contributes to the complex "histone code" that regulates DNA accessibility .

This modification has been implicated in several critical nuclear processes including transcriptional regulation, DNA damage response pathways, and apoptotic chromatin condensation. H2BS14p works in concert with other histone modifications to create specific binding platforms for chromatin-remodeling enzymes and transcription factors, thereby influencing gene expression programs and chromatin architecture .

How does H2B S14 phosphorylation differ from other H2B modifications such as Y121 phosphorylation?

While both are post-translational modifications of histone H2B, they serve distinct regulatory functions in chromatin dynamics. H2B S14 phosphorylation occurs at a serine residue and has been predominantly associated with apoptotic processes and DNA damage responses. In contrast, H2B Y121 phosphorylation occurs at a tyrosine residue and functions primarily as a regulatory switch during eukaryotic transcription .

Research has shown that Y121 phosphorylation status directly influences H2B ubiquitination at lysine 120 (K120), with dephosphorylation of Y121 by phosphatases like SHP-1 promoting efficient H2B K120 ubiquitination . This demonstrates how different modifications on the same histone protein can participate in distinct regulatory pathways within the nucleus, highlighting the complexity of the histone code in chromatin regulation .

What are the optimal sample preparation techniques for detecting H2B S14 phosphorylation in cell lines?

For optimal detection of H2B S14 phosphorylation in cell lines, acid extraction has proven to be an effective sample preparation method. This approach efficiently extracts histones while preserving their post-translational modifications. As demonstrated in Western blot applications, acid extracts from HeLa cells have been successfully used to detect H2BS14p using antibodies like the Rabbit Recombinant Monoclonal H2B phospho S14 antibody .

The procedure involves:

• Harvesting cells and washing with cold PBS

• Lysing cells in hypotonic buffer containing histone deacetylase and phosphatase inhibitors

• Extracting histones with 0.2N HCl

• Neutralizing with appropriate buffer

• Quantifying protein concentration prior to analysis

This method maximizes the signal-to-noise ratio and ensures specific detection of the phosphorylated form of H2B at serine 14 while minimizing artifacts that might arise from improper sample handling .

How can researchers validate the specificity of Phospho-HIST1H2BC (S14) antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable experimental results. For Phospho-HIST1H2BC (S14) antibodies, a comprehensive validation approach should include:

• Peptide competition assays: Pre-incubating the antibody with the phospho-specific peptide should abolish signal detection, while pre-incubation with non-phosphorylated peptide should not affect antibody recognition

• Phosphatase treatment: Treating samples with λ-phosphatase should eliminate the phospho-specific signal while preserving the total H2B signal detected with a pan-H2B antibody

• Use of phospho-mimetic mutants: Comparing antibody reactivity against wild-type H2B versus S14A (non-phosphorylatable) and S14E (phospho-mimetic) mutants can verify specificity

• Cross-reactivity assessment: Testing the antibody against other histones or phosphorylated proteins to ensure it specifically detects H2B phosphorylated at S14 and not other phosphorylated residues

These validation steps are analogous to those used for other phospho-specific histone antibodies, such as the approach described for validating H2B Y121 phospho-specific antibodies .

How can ChIP-seq be optimized for genome-wide mapping of H2BS14p during DNA damage response?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for mapping H2BS14p during DNA damage response requires careful optimization to achieve high-quality, reliable results:

• Crosslinking optimization: Dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde can improve retention of phosphorylated histones and associated proteins

• Sonication parameters: Adjust sonication conditions to generate chromatin fragments of 200-500bp while preserving phospho-epitopes

• Antibody selection and validation: Use highly specific Phospho-HIST1H2BC (S14) antibodies validated for ChIP applications (specificity can be verified using approaches outlined in section 2.2)

• Input controls: Generate proper input controls from the same chromatin preparation prior to immunoprecipitation

• Spike-in normalization: Implement spike-in controls using chromatin from a different species to normalize for technical variations between experimental conditions

• Data analysis considerations: Apply specialized peak-calling algorithms designed for histone modifications that typically form broad domains rather than sharp peaks

These optimization steps are particularly important given the potential challenges in generating ChIP-grade antibodies for phospho-epitopes, similar to limitations noted with SHP-1 antibodies for ChIP applications in histone phosphorylation studies .

What is the interplay between H2B S14 phosphorylation and other histone modifications in the DNA damage response pathway?

H2B S14 phosphorylation functions within a complex network of histone modifications during DNA damage response. This interplay creates a dynamic chromatin environment that facilitates repair processes:

• Sequential modification cascades: H2B S14 phosphorylation often occurs in coordination with H2A.X S139 phosphorylation (γH2A.X), with the latter serving as an initial DNA damage marker and the former contributing to subsequent chromatin remodeling steps

• Cross-talk with histone ubiquitination: While Y121 phosphorylation status affects H2B K120 ubiquitination during transcription , S14 phosphorylation may similarly influence ubiquitination events during DNA repair processes

• Coordination with histone acetylation: H2B S14 phosphorylation works in concert with acetylation of H3 and H4 to create accessible chromatin regions for repair machinery

• Methylation-phosphorylation switches: The phosphorylation status of H2B S14 can influence methylation patterns on other histones, particularly H3K4 and H3K79, creating feedback loops that regulate the progression of repair

This complex regulatory network ensures that chromatin structure is appropriately modified to facilitate repair while maintaining genomic integrity .

What are common technical challenges when using Phospho-HIST1H2BC (S14) antibodies in immunofluorescence studies?

Researchers may encounter several challenges when using Phospho-HIST1H2BC (S14) antibodies for immunofluorescence studies:

• Fixation artifacts: Overfixation can mask epitopes while underfixation may compromise nuclear structure. Optimization of fixation time (typically 10-15 minutes with 4% paraformaldehyde) is essential

• Permeabilization conditions: The nuclear localization of histones requires effective permeabilization without disrupting nuclear architecture. A sequential approach using 0.2% Triton X-100 followed by 0.5% SDS treatment can improve antibody accessibility to chromatin-bound epitopes

• Background fluorescence: Non-specific binding can obscure true signals. Implement extended blocking steps (minimum 1 hour) with 5% BSA or 10% normal serum from the same species as the secondary antibody

• Signal intensity variations: Phosphorylation levels may vary depending on cell cycle phase or stress conditions. Synchronize cells when studying cell cycle-dependent phosphorylation events

• Co-visualization challenges: When performing co-localization studies with other nuclear markers, carefully select fluorophores with minimal spectral overlap

Successful immunofluorescence studies have been conducted using antibodies like ab222762 at 2 μg/ml concentration in HeLa cells, demonstrating nuclear localization patterns of H2BS14p .

How can researchers distinguish between specific and non-specific signals when detecting H2BS14p by Western blot?

Distinguishing between specific and non-specific signals requires rigorous controls and optimized protocols:

• Lysate preparation optimization:

  • Use acid extraction methods specifically designed for histones

  • Include phosphatase inhibitors in all buffers to preserve phosphorylation status

  • Use freshly prepared samples to minimize degradation

• Critical controls:

  • Include λ-phosphatase-treated samples as negative controls

  • Use recombinant H2B proteins (phosphorylated and non-phosphorylated) as reference standards

  • Include samples from cells treated with kinase inhibitors that target pathways known to phosphorylate H2B

• Blotting parameters:

  • Use PVDF membranes for better retention of phospho-proteins

  • Optimize antibody concentration (typically starting at 0.5 μg/mL for antibodies like ab222762)

  • Extend blocking time to minimize background (minimum 1 hour at room temperature)

  • Include 0.1% Tween-20 in wash buffers to reduce non-specific binding

• Signal verification:

  • Confirm band size matches histone H2B (~14-15 kDa)

  • Verify signal disappears in peptide competition assays

  • Compare signal patterns with published literature

Following these guidelines will help ensure that the detected signals genuinely represent H2BS14p rather than artifacts or cross-reactivity with other phosphorylated proteins.

How does H2B S14 phosphorylation contribute to transcriptional regulation compared to other phosphorylation sites like Y121?

While Y121 phosphorylation has been established as a regulatory switch during transcription with its dephosphorylation promoting H2B ubiquitination , the role of S14 phosphorylation in transcriptional regulation presents a complex and evolving research area:

• Context-dependent effects: H2B S14 phosphorylation can either activate or repress transcription depending on genomic context and the presence of other histone modifications

• Recruitment mechanisms: Unlike Y121 phosphorylation that interacts with the Paf1 complex during transcription elongation , S14 phosphorylation may recruit different effector proteins such as chromatin remodelers or repressive complexes

• Gene-specific regulation: S14 phosphorylation may exhibit gene-specific effects, potentially regulating subsets of genes involved in stress responses or apoptotic pathways, in contrast to the more general transcriptional role of Y121 phosphorylation

• Kinase/phosphatase regulation: While SHP-1 has been identified as the phosphatase that dephosphorylates Y121 , the kinases and phosphatases that regulate S14 phosphorylation during transcription remain subjects of ongoing investigation

These comparative studies between different phosphorylation sites on the same histone provide important insights into the nuanced "histone code" that governs transcriptional regulation .

What is the potential role of H2BS14p in neurodegenerative diseases and cancer progression?

Emerging research suggests H2BS14p may play significant roles in both neurodegenerative diseases and cancer:

• Neurodegenerative diseases:

  • Aberrant H2BS14p levels have been observed in brain tissues from neurodegenerative disease models

  • Dysregulation of H2BS14p may contribute to neuronal apoptosis in conditions like Alzheimer's and Parkinson's diseases

  • The modification may influence expression of neuroprotective genes under stress conditions

• Cancer progression:

  • Altered H2BS14p patterns have been detected in various cancer types

  • Changes in H2BS14p may contribute to genomic instability through impaired DNA damage responses

  • Cancer cells might exploit H2BS14p-dependent pathways to evade apoptosis

• Therapeutic implications:

  • Targeting enzymes that regulate H2BS14p could represent a novel therapeutic approach

  • H2BS14p levels might serve as biomarkers for disease progression or treatment response

  • Combination therapies targeting multiple histone modifications including H2BS14p could enhance therapeutic efficacy

This represents an exciting frontier in epigenetic research, with potential translational applications in disease diagnosis and treatment strategies.

What are the recommended protocols for studying dynamic changes in H2BS14p during apoptosis using flow cytometry?

A comprehensive flow cytometry protocol for analyzing H2BS14p during apoptosis should include:

• Sample preparation:

  • Induce apoptosis using appropriate stimuli (e.g., staurosporine, UV irradiation)

  • Collect cells at multiple time points (0, 1, 3, 6, 12, 24 hours)

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

  • Permeabilize with 90% methanol for 30 minutes on ice

• Antibody staining:

  • Block with 5% BSA in PBS for 30 minutes

  • Incubate with Phospho-HIST1H2BC (S14) antibody (1:100 dilution)

  • Co-stain with markers of apoptosis (e.g., cleaved caspase-3)

  • Include appropriate isotype controls

• Multiparameter analysis:

  • Gate on single cells using forward/side scatter properties

  • Analyze H2BS14p signal intensity in relation to apoptotic markers

  • Perform cell cycle analysis using DNA content staining (propidium iodide)

• Controls and validation:

  • Include phosphatase-treated samples as negative controls

  • Use caspase inhibitors to block apoptosis as functional controls

  • Validate key findings using complementary techniques (Western blot, immunofluorescence)

This approach allows quantitative assessment of H2BS14p dynamics during apoptosis at the single-cell level, providing insights into the temporal relationship between H2BS14p and other apoptotic events.

How should researchers design ChIP-qPCR experiments to study H2BS14p enrichment at specific genomic loci?

Designing effective ChIP-qPCR experiments for H2BS14p requires attention to several key factors:

• Experimental design:

  • Include biological replicates (minimum n=3)

  • Incorporate appropriate treatment conditions (DNA damage inducers, apoptotic stimuli)

  • Establish time courses to capture dynamic changes

• Sample processing:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Sonicate chromatin to 200-500bp fragments

  • Pre-clear chromatin with protein A/G beads

  • Perform immunoprecipitation with Phospho-HIST1H2BC (S14) antibody

• Controls:

  • Include IgG negative control

  • Use pan-H2B antibody as reference for total H2B occupancy

  • Include positive control loci known to be enriched for H2BS14p

  • Include negative control regions like gene deserts

• Primer design for qPCR:

  • Design primers for regions of interest (promoters, enhancers, gene bodies)

  • Ensure primers generate 80-150bp amplicons

  • Verify primer specificity and efficiency

  • Include primer sets for normalization regions

• Data analysis:

  • Calculate enrichment using percent input or fold enrichment over IgG

  • Normalize H2BS14p signal to total H2B when appropriate

  • Apply statistical tests to determine significance

This approach can be modeled after ChIP-qPCR protocols used successfully for studying other histone modifications, such as those employed to analyze SHP-1 and H2B Y121 phosphorylation at gene loci .

How do polyclonal and monoclonal Phospho-HIST1H2BC (S14) antibodies compare in terms of specificity and applications?

Polyclonal and monoclonal Phospho-HIST1H2BC (S14) antibodies have distinct characteristics that influence their performance across different applications:

When selecting between these antibody types, researchers should consider their specific experimental requirements, balancing sensitivity, specificity, and application compatibility .

What are the key considerations when comparing different commercial Phospho-HIST1H2BC (S14) antibodies for research applications?

Researchers should evaluate multiple factors when selecting commercial Phospho-HIST1H2BC (S14) antibodies:

• Validation data comprehensiveness:

  • Availability of specificity tests (peptide competition, phosphatase treatment)

  • Validation across multiple techniques (WB, IF, ChIP)

  • Testing in relevant cell types or tissues

• Technical specifications:

  • Antibody format (whole IgG, Fab fragments)

  • Storage buffer composition and stability (e.g., PBS with 50% glycerol and 0.03% Proclin 300)

  • Working concentration ranges for different applications

• Production details:

  • Immunogen design and carrier protein

  • Host species compatibility with experimental system

  • Purification method (e.g., antigen affinity chromatography)

• Application-specific performance:

  • Signal-to-noise ratio in intended application

  • Required sample types (acid extracts, fixed cells)

  • Detection limits and dynamic range

• Support and reproducibility:

  • Lot-to-lot consistency data

  • Customer support for troubleshooting

  • Publication record with the antibody

By systematically evaluating these factors, researchers can select the most appropriate antibody for their specific experimental needs, whether using polyclonal antibodies like the Invitrogen product or monoclonal antibodies like ab222762 .

How might single-cell epigenomic approaches advance our understanding of H2BS14p dynamics in heterogeneous cell populations?

Single-cell epigenomic technologies represent a frontier in understanding H2BS14p dynamics:

• Technology integration opportunities:

  • Adapting CUT&Tag or CUT&RUN protocols for single-cell analysis of H2BS14p distribution

  • Combining single-cell RNA-seq with H2BS14p profiling to correlate modification status with transcriptional output

  • Implementing mass cytometry (CyTOF) with phospho-specific antibodies to simultaneously analyze H2BS14p alongside other cellular markers

• Biological insights possible through single-cell approaches:

  • Resolving cell-to-cell variability in H2BS14p during stress responses or development

  • Identifying rare cell populations with unique H2BS14p signatures

  • Tracking temporal dynamics of H2BS14p in individual cells during differentiation or disease progression

• Technical challenges to address:

  • Improving antibody specificity for single-cell applications

  • Developing computational methods to integrate H2BS14p data with other single-cell modalities

  • Minimizing technical noise while preserving biological variation

These approaches could provide unprecedented insights into how H2BS14p contributes to cellular heterogeneity and epigenetic regulation in complex tissues and disease states.

What are emerging approaches for studying the enzymes responsible for regulating H2BS14 phosphorylation status?

Innovative approaches for identifying and characterizing H2BS14p regulatory enzymes include:

• Screening strategies:

  • CRISPR-Cas9 screens targeting known kinases and phosphatases to identify regulators of H2BS14p

  • Chemical genetic approaches using analog-sensitive kinases to identify direct phosphorylation events

  • Proximity labeling techniques to identify proteins associating with H2BS14p regions

• Structural biology approaches:

  • Cryo-EM studies of regulatory enzyme complexes with nucleosomes containing H2BS14p

  • Hydrogen-deuterium exchange mass spectrometry to map enzyme-substrate interactions

  • Computational modeling to predict binding interfaces between histones and regulatory enzymes

• In vivo studies:

  • Development of genetically encoded biosensors for real-time visualization of H2BS14p dynamics

  • Animal models with mutations in H2B S14 or its regulatory enzymes

  • Patient-derived samples to study dysregulation in human disease contexts

These approaches build upon methodologies successfully employed for other histone modifications, such as those used to identify SHP-1 as a regulator of H2B Y121 phosphorylation , and will help elucidate the complete regulatory network controlling H2BS14p in various biological contexts.