Phospho-Histone H3.1 (S1) Recombinant Monoclonal Antibody

What is Histone H3.1 and what biological significance does S1 phosphorylation have?

Histone H3.1 is one of the core components of the nucleosome, the basic unit of chromatin. It belongs to the H3 family of histone proteins and differs from the H3.3 variant by specific amino acid residues. H3.1 is primarily expressed during S-phase and incorporated into chromatin during DNA replication, making it a replication-coupled (RC) histone .

Phosphorylation at serine 1 (S1) of histone H3.1 is associated with:

• Promotion of chromatin condensation leading to gene repression

• Regulation of chromosome condensation during mitosis

• Involvement in DNA damage response and repair mechanisms

• Contribution to cell cycle regulation and epigenetic signaling

Unlike other phosphorylation sites such as S10 or S28, the S1 phosphorylation appears to be more directly involved in transcriptional silencing rather than activation.

How do recombinant monoclonal antibodies differ from traditional monoclonal antibodies for histone research?

Recombinant monoclonal antibodies offer several advantages over traditional hybridoma-derived antibodies:

For histone research specifically, recombinant antibodies can be designed to recognize precise post-translational modifications with minimal cross-reactivity to other histone marks, which is critical when studying the nuanced epigenetic code .

What are the recommended applications and dilutions for Phospho-Histone H3.1 (S1) antibodies?

Based on manufacturer recommendations across multiple providers:

Optimization is essential as detection sensitivity may vary depending on:

• Cell type or tissue used

• Fixation and permeabilization methods

• Extent of S1 phosphorylation in your experimental system

• Secondary antibody and detection system employed

How can researchers validate the specificity of Phospho-Histone H3.1 (S1) antibodies?

Rigorous validation of phospho-specific antibodies is crucial to ensure experimental accuracy:

• Peptide competition assay: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides corresponding to the target epitope. A specific signal should be blocked only by the phosphorylated peptide.

• Phosphatase treatment controls: Treat one sample with lambda phosphatase to remove phosphorylation and compare to untreated samples. The signal should disappear in phosphatase-treated samples.

• Knock-in/knock-out validation: Use cells where the target site has been mutated (S1A) to prevent phosphorylation or use histone H3.1 knockout models as negative controls.

• Kinase inhibition: Use specific kinase inhibitors that block the phosphorylation of the S1 residue to confirm specificity.

• Orthogonal techniques: Confirm results using alternative methods such as mass spectrometry to detect the phosphorylation state of H3.1 S1.

• Positive controls: Include samples known to have high levels of H3.1 S1 phosphorylation, such as cells synchronized at specific cell cycle stages .

What cell culture and sample preparation methods maximize detection of H3.1 S1 phosphorylation?

Optimal detection of H3.1 S1 phosphorylation requires careful sample preparation:

Cell Culture Considerations:

• Synchronization: Use techniques like thymidine block combined with nocodazole to enrich for mitotic cells when H3.1 phosphorylation is often elevated

• Stress induction: Various cellular stresses can alter histone phosphorylation patterns

• Inhibitor treatments: Calyculin A (phosphatase inhibitor) can be used to preserve phosphorylation states

Sample Preparation for Western Blot:

• Add phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) to all buffers

• Use acid extraction methods for histone isolation to preserve phosphorylation marks

• Keep samples cold during processing to minimize phosphatase activity

• Consider direct lysis in SDS sample buffer for rapid fixation of phosphorylation states

For Immunofluorescence:

• Quick fixation with 4% paraformaldehyde is often preferred

• Brief permeabilization with 0.1-0.2% Triton X-100

• Include phosphatase inhibitors in buffers

• Consider antigen retrieval methods if necessary

What are the technical differences between detecting H3.1 S1 phosphorylation versus other histone H3 phosphorylation marks?

Different histone H3 phosphorylation sites present unique technical challenges:

For multiplex detection of different modifications:

• Consider sequential detection with appropriate stripping between antibodies

• Use fluorescent multiplex Western blotting with differently labeled secondary antibodies

• For imaging applications, select antibodies from different host species to allow simultaneous detection

How does Histone H3.1 S1 phosphorylation differ functionally from the more well-characterized S10 phosphorylation?

These phosphorylation marks have distinct functions and dynamics in cellular processes:

The spatial and temporal dynamics of these marks reflect their distinct roles in chromatin biology. While S10 phosphorylation is a robust marker of mitosis widely used in cell cycle studies, S1 phosphorylation appears to play more subtle roles in transcriptional regulation and chromatin organization .

What experimental approaches can be used to study the kinases responsible for H3.1 S1 phosphorylation?

Several complementary approaches can identify and characterize kinases that phosphorylate H3.1 at S1:

• In vitro kinase assays:

  • Incubate purified recombinant H3.1 with candidate kinases

  • Detect phosphorylation using Phospho-H3.1 (S1) antibodies

  • Quantify using Western blot or ELISA-based methods

  • Can be performed with γ-32P-ATP for radiometric analysis

• Kinase inhibitor screens:

  • Treat cells with panels of specific kinase inhibitors

  • Monitor changes in H3.1 S1 phosphorylation levels

  • Create dose-response curves to identify potential kinases

• Genetic approaches:

  • CRISPR/Cas9 knockout or knockdown of candidate kinases

  • Overexpression of constitutively active or dominant negative kinase mutants

  • Monitor effects on global H3.1 S1 phosphorylation levels

• Immunoprecipitation-based methods:

  • Co-immunoprecipitation to identify kinases physically associated with H3.1

  • Phosphorylation of immunoprecipitated H3.1 using cell extracts

  • Mass spectrometry to identify kinases in complex with H3.1

• HTRF (Homogeneous Time-Resolved Fluorescence) assays:

  • Enables high-throughput screening of kinase activity

  • Uses donor and acceptor fluorophore-labeled antibodies

  • Signal intensity correlates with phosphorylation levels

How can researchers distinguish between H3.1 and H3.3 phosphorylation patterns in experimental systems?

Distinguishing between H3 variants requires specialized techniques due to their high sequence similarity:

• Variant-specific antibodies:

  • Use antibodies that recognize the unique regions of H3.1 or H3.3

  • H3.3 S31 phosphorylation is a variant-specific mark (not present in H3.1)

  • For H3.1, antibodies targeting regions containing A31 (vs S31 in H3.3) can be used

• Sequential chromatin immunoprecipitation (ChIP):

  • First IP with variant-specific antibody

  • Second IP with phosphorylation-specific antibody

  • Identifies genomic regions containing specific variant with specific modifications

• Mass spectrometry approaches:

  • Can distinguish variant-specific peptides and their modifications

  • Enables quantitative analysis of modification stoichiometry

  • Can detect combinatorial modifications not possible with antibodies

• Stable isotope labeling by amino acids in cell culture (SILAC):

  • Label histones during protein synthesis

  • Monitor turnover rates of different variants and their modifications

  • Particularly useful for studying replication-dependent vs. independent incorporation

• Exogenous tagged histones:

  • Express tagged versions of H3.1 or H3.3 (HA, FLAG, GFP)

  • Can be immunoprecipitated with tag-specific antibodies

  • Monitor phosphorylation on specific variant using phospho-specific antibodies

What is the relationship between H3.1 S1 phosphorylation and other histone modifications in the context of the histone code?

H3.1 S1 phosphorylation functions within the complex network of histone modifications that constitute the histone code:

• Cross-talk with adjacent modifications:

  • S1 phosphorylation may prevent methylation or acetylation of neighboring residues (K4, R2)

  • Can influence reader protein binding to nearby modified residues

  • May alter accessibility of the histone tail to other modifying enzymes

• Combinatorial effects with other modifications:

  • The combination of S1ph with specific lysine methylation states (K4me3, K9me3, K27me3) may signal for distinct functional outcomes

  • Sequential establishment of modifications during cell cycle progression creates specific chromatin states

• Experimental approaches to study cross-talk:

  • Use recombinant histones with defined modifications for in vitro studies

  • Mass spectrometry to identify co-occurring modifications

  • Sequential ChIP to identify genome regions with multiple modifications

  • Antibodies specifically recognizing combinatorial modifications

• Functional consequences:

  • S1 phosphorylation may influence the binding of chromatin remodeling complexes

  • Can affect higher-order chromatin structure and accessibility

  • May target specific genomic regions for silencing or activation depending on context

• Cell cycle dynamics:

  • Progressive, coordinated restoration of histone modifications occurs after DNA replication

  • S1 phosphorylation shows distinct temporal patterns compared to other modifications such as S10 phosphorylation

What are common technical issues when working with Phospho-Histone H3.1 (S1) antibodies and how can they be addressed?

Researchers frequently encounter these challenges when detecting H3.1 S1 phosphorylation:

For Western blotting specifically:

• Transfer efficiency: Use transfer methods optimized for low molecular weight proteins

• Loading controls: Consider using total H3 antibodies or non-histone loading controls

• Quantification: Normalize phospho-signal to total H3 signal for accurate comparisons

How can researchers optimize detection of phosphorylated H3.1 in fixed tissue samples for immunohistochemistry?

Optimizing phospho-histone detection in tissue samples requires special considerations:

• Fixation protocols:

  • Overfixation can mask epitopes; consider shorter fixation times (4-24 hours)

  • Freshly prepared 4% paraformaldehyde is often optimal

  • Avoid acidic fixatives that may affect phospho-epitopes

  • Consider perfusion fixation for animal tissues when possible

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Optimize time and temperature (typically 95-100°C for 15-30 minutes)

  • Allow slow cooling to room temperature

  • Include phosphatase inhibitors in retrieval buffers

• Signal amplification:

  • Consider tyramide signal amplification systems

  • Polymer-based detection systems may provide better sensitivity

  • Avoid excessive amplification that can increase background

• Controls and validation:

  • Include phosphatase-treated sections as negative controls

  • Use tissues known to contain cells with high H3.1 S1 phosphorylation (e.g., proliferating tissues)

  • Consider mitotic cells (identifiable by DAPI) as internal positive controls

• Quantification approaches:

  • Digital image analysis can provide quantitative data

  • Consider normalization to total H3 staining

  • Set consistent thresholds for positive staining

What emerging technologies offer improved detection of histone phosphorylation beyond traditional methods?

Several advanced technologies are enhancing histone phosphorylation research:

• Single-cell analysis approaches:

  • Mass cytometry (CyTOF) for multiparameter analysis of histone modifications

  • Single-cell Western blotting for phospho-histone analysis

  • Imaging mass spectrometry for spatial distribution of modifications

• High-sensitivity detection methods:

  • HTRF (Homogeneous Time-Resolved Fluorescence) assays provide plate-based detection without Western blotting

  • Luminex bead-based multiplex assays for simultaneous detection of multiple phosphorylation sites

  • Digital ELISA platforms offering femtomolar sensitivity

• Genomic approaches:

  • CUT&RUN and CUT&Tag methods for improved chromatin profiling

  • ChIP-seq combining phospho-specific antibodies with next-generation sequencing

  • CHIC/HiChIP methods combining chromatin conformation with histone modification analysis

• Live-cell imaging techniques:

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

  • Specific fluorescent probes for visualizing phosphorylated histones

  • Optogenetic tools to manipulate kinase activity with spatial and temporal precision

• Synthetic biology approaches:

  • Engineered histones with unnatural amino acids at specific positions

  • Chemical methods for generating homogeneously modified histones

  • Designer reader domains for detecting specific modification patterns

How is H3.1 S1 phosphorylation dysregulated in cancer and other diseases?

While H3.1 S1 phosphorylation is less studied than other histone marks in disease contexts, emerging research suggests several important connections:

• Cancer associations:

  • Altered H3.1 S1 phosphorylation patterns observed in several cancer types

  • May contribute to aberrant gene silencing of tumor suppressors

  • Dysregulation of kinases responsible for S1 phosphorylation often occurs in tumors

  • Potential biomarker for specific cancer subtypes or progression stages

• Neurodegenerative disorders:

  • Altered histone phosphorylation patterns observed in models of neurodegeneration

  • May contribute to transcriptional dysregulation in affected neurons

  • Could represent potential therapeutic targets

• Inflammatory conditions:

  • Inflammation-associated signaling can affect histone phosphorylation patterns

  • May contribute to epigenetic reprogramming during chronic inflammation

• Developmental disorders:

  • Proper histone modification dynamics are essential for development

  • Mutations affecting histone H3.1 or its modifying enzymes linked to developmental abnormalities

• Experimental approaches:

  • Tissue microarrays with phospho-H3.1 (S1) immunostaining to assess clinical correlations

  • Genetic models manipulating kinases/phosphatases regulating S1 phosphorylation

  • Integration with other epigenetic markers for comprehensive disease profiling

What are the technical considerations for studying H3.1 S1 phosphorylation in patient-derived samples?

Working with clinical specimens presents unique challenges for histone phosphorylation analysis:

• Sample preservation:

  • Phosphorylation marks are labile and sensitive to post-collection handling

  • Flash freezing or immediate fixation is optimal

  • Document ischemia time and preservation method

  • Consider phosphatase inhibitors during sample collection

• FFPE tissue considerations:

  • Formalin fixation can affect epitope accessibility

  • Standardize fixation protocols (time, buffer, temperature)

  • Optimize antigen retrieval methods specifically for phospho-epitopes

  • Consider using phosphatase inhibitors in retrieval buffers

• Controls and normalization:

  • Use matched normal tissues when possible

  • Consider adjacent normal tissue within samples as internal control

  • Normalize phospho-signal to total H3 levels

  • Include phosphatase-treated sections as technical controls

• Quantification methods:

  • Digital pathology approaches for objective quantification

  • Consider H-score methods (intensity × percentage positive cells)

  • Blinded scoring by multiple observers

  • Use image analysis software for consistent threshold application

• Integration with other biomarkers:

  • Multiplex immunohistochemistry/immunofluorescence for co-localization studies

  • Correlate with clinical parameters and outcomes

  • Consider laser capture microdissection for cell type-specific analysis