Phospho-HIST1H1E (S35) Antibody

What is HIST1H1E and why is phosphorylation at S35 significant?

HIST1H1E (Histone H1.4) is a linker histone that binds to DNA between nucleosomes to form higher-order chromatin structures. Phosphorylation at serine 35 (S35) is a post-translational modification that plays a crucial role in regulating chromatin dynamics, particularly during cell cycle progression and transcriptional regulation. The phosphorylation status at S35 has been linked to changes in chromatin compaction and accessibility for transcription factors, making it an important epigenetic marker for studies of gene expression regulation .

What are the recommended applications for Phospho-HIST1H1E (S35) antibody?

The Phospho-HIST1H1E (S35) antibody is validated for multiple applications including:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• ICC (Immunocytochemistry)

• IF (Immunofluorescence)

Recommended dilutions for optimal results are:

• ICC: 1:20-1:200

• IF: 1:50-1:200

These applications allow researchers to detect and quantify phosphorylated H1.4 in various experimental settings, providing insights into epigenetic regulation mechanisms .

What is the specificity of the Phospho-HIST1H1E (S35) antibody?

This polyclonal antibody is generated against a peptide sequence surrounding the phosphorylated serine 35 site of human Histone H1.4 (UniProt ID: P10412). It specifically recognizes the phosphorylated form of HIST1H1E at Ser35, enabling researchers to distinguish between the phosphorylated and non-phosphorylated states of the protein. This specificity is critical for studies investigating the functional consequences of this post-translational modification in various cellular processes .

How should I design an immunofluorescence experiment using Phospho-HIST1H1E (S35) antibody?

For optimal immunofluorescence results with Phospho-HIST1H1E (S35) antibody:

• Sample preparation:

  • Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilization: 0.2% Triton X-100 for 10 minutes

  • Blocking: 5% BSA in PBS for 1 hour

• Antibody incubation:

  • Primary antibody: Use Phospho-HIST1H1E (S35) at 1:50-1:200 dilution overnight at 4°C

  • Secondary antibody: Anti-rabbit IgG conjugated with fluorophore at 1:500 dilution for 1 hour at room temperature

• Controls to include:

  • Negative control: Secondary antibody only

  • Dephosphorylation control: Treat samples with phosphatase before antibody incubation

  • Cell cycle synchronization: Compare G1, S, and G2/M phases as phosphorylation levels may vary

• Counterstaining:

  • DAPI for nuclear visualization

  • Additional markers for cell cycle or chromatin states as needed for co-localization studies

What are effective sample preparation protocols for nuclear protein extraction when studying HIST1H1E?

Effective nuclear protein extraction for HIST1H1E studies requires careful fractionation to maintain phosphorylation status:

• Cell harvesting and lysis:

  • Collect cells by gentle trypsinization

  • Wash with ice-cold PBS containing phosphatase inhibitors (10mM NaF, 1mM Na3VO4)

  • Resuspend in hypotonic buffer (10mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl2, 0.34M sucrose, 10% glycerol, 1mM DTT, protease and phosphatase inhibitors)

  • Add Triton X-100 to 0.1% final concentration

  • Incubate on ice for 8 minutes

• Nuclear isolation:

  • Centrifuge at 1,300×g for 5 minutes at 4°C

  • Wash nuclear pellet once with hypotonic buffer

  • Resuspend in high-salt extraction buffer (20mM HEPES pH 7.9, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 25% glycerol, protease and phosphatase inhibitors)

  • Incubate on ice for 30 minutes with periodic vortexing

• Quality control:

  • Assess nuclear purity by Western blot using markers like Lamin B (nuclear) and GAPDH (cytoplasmic)

  • Ensure minimal mitochondrial contamination by checking for mitochondrial markers

This protocol helps preserve the phosphorylation status of HIST1H1E for subsequent analysis while minimizing contamination from other cellular compartments.

How can I optimize ChIP protocols when using Phospho-HIST1H1E (S35) antibody?

Optimizing ChIP for Phospho-HIST1H1E (S35) requires special considerations:

• Crosslinking optimization:

  • Use 1% formaldehyde for 10 minutes at room temperature

  • For linker histones, consider dual crosslinking with 1.5mM EGS (ethylene glycol bis[succinimidylsuccinate]) for 30 minutes before formaldehyde

• Chromatin fragmentation:

  • Sonicate to generate fragments of 200-500bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 3-5μg of Phospho-HIST1H1E (S35) antibody per 25μg of chromatin

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

  • Extended incubation (overnight at 4°C) with gentle rotation

• Washing stringency:

  • Perform sequential washes with increasing salt concentration

  • Include a LiCl wash to reduce non-specific binding

• Controls:

  • Input chromatin (10%)

  • IgG negative control

  • Positive control using antibody against unmodified HIST1H1E

• DNA purification and analysis:

  • Purify DNA using phenol-chloroform extraction or commercial kits

  • Analyze enrichment by qPCR targeting regions known to be associated with H1.4

How do I interpret changes in Phospho-HIST1H1E (S35) signal during cell cycle progression?

Phosphorylation of HIST1H1E at S35 exhibits distinct patterns throughout the cell cycle:

When interpreting changes in Phospho-HIST1H1E (S35) signals:

• Temporal context: Always consider the cell cycle stage of your samples

• Spatial distribution: Note whether signals are broadly nuclear or localized to specific domains

• Co-localization analysis: Compare with other cell cycle markers (e.g., Ki67, PCNA)

• Quantitative assessment: Measure signal intensity relative to total HIST1H1E levels

• Perturbation responses: Evaluate how treatments affecting CDKs or other kinases alter the phosphorylation pattern

What are common causes of non-specific binding when using Phospho-HIST1H1E (S35) antibody and how can they be addressed?

Non-specific binding issues can compromise data quality when working with phospho-specific antibodies:

For best results, always run appropriate controls, including:

• Phosphatase-treated samples

• Competitive blocking with phosphopeptide

• Validation with alternative detection methods

How can I resolve contradictory results between phospho-specific antibody detection and mass spectrometry quantification of HIST1H1E phosphorylation?

When faced with discrepancies between antibody-based detection and mass spectrometry (MS) results for Phospho-HIST1H1E (S35):

• Analytical considerations:

  • Antibody specificity: Verify using phospho-null mutants (S35A) and phospho-mimetic mutants (S35D/E)

  • MS sample preparation: Ensure preservation of phosphorylation using appropriate extraction buffers with phosphatase inhibitors

  • MS ionization efficiency: Phosphopeptides often have lower ionization efficiency; consider phospho-enrichment steps

• Biological variables:

  • Dynamic range: MS may not detect low-abundance phosphorylation that antibodies can detect

  • Epitope accessibility: In some contexts, the S35 phosphorylation site may be masked in complexes

  • Localized vs. global changes: Antibody-based imaging detects localized changes that may be diluted in whole-cell MS samples

• Resolution strategies:

  • Perform fractionation to enrich for HIST1H1E before MS analysis

  • Use parallel reaction monitoring (PRM) for targeted MS quantification

  • Apply phosphopeptide enrichment (TiO2 or IMAC) before MS

  • Validate with orthogonal methods like Phos-tag gels

  • Control for potential confounding factors like cell cycle synchronization

How can Phospho-HIST1H1E (S35) antibody be used to investigate the relationship between histone phosphorylation and chromatin remodeling?

Investigating the relationship between HIST1H1E phosphorylation and chromatin remodeling requires multi-modal approaches:

• Chromatin accessibility analysis:

  • Combine Phospho-HIST1H1E (S35) ChIP-seq with ATAC-seq or DNase-seq

  • Compare phosphorylated vs. non-phosphorylated HIST1H1E distribution relative to accessible regions

  • Analyze temporal changes following signaling pathway activation

• Nucleosome dynamics assessment:

  • Perform MNase-seq in conjunction with Phospho-HIST1H1E (S35) ChIP

  • Map nucleosome positioning changes relative to phosphorylation status

  • Use live-cell imaging with fluorescently tagged HIST1H1E phospho-mimetics

• Protein interaction studies:

  • Conduct Phospho-HIST1H1E (S35) antibody-based co-IP followed by mass spectrometry

  • Identify differential interactors between phosphorylated and non-phosphorylated forms

  • Validate key interactions using proximity ligation assays

• Functional genomics approaches:

  • Compare transcriptome changes (RNA-seq) with Phospho-HIST1H1E (S35) ChIP-seq

  • Implement CRISPR-based histone mutagenesis (S35A, S35D) to assess functional consequences

  • Analyze effects of kinase or phosphatase inhibitors on chromatin landscapes

What are the methodological considerations for studying the role of Phospho-HIST1H1E (S35) in development and differentiation?

Studying Phospho-HIST1H1E (S35) in development and differentiation requires specialized approaches:

• Temporal sampling strategy:

  • Define critical developmental timepoints for analysis

  • Consider both rapid transitions (hours) and long-term changes (days)

  • Include matched controls at each timepoint

• Cell heterogeneity management:

  • Implement single-cell approaches (CyTOF, scRNA-seq with protein detection)

  • Use FACS to isolate pure populations based on differentiation markers

  • Apply tissue-specific markers for in vivo studies

• Quantification methods:

  • Develop ratiometric imaging approaches (Phospho-HIST1H1E/total HIST1H1E)

  • Use in situ proximity ligation assays for tissue samples

  • Apply phospho-flow cytometry for quantitative population analysis

• Perturbation strategies:

  • Design stage-specific inhibition of relevant kinases/phosphatases

  • Create conditional phospho-mutant models using CRISPR/Cas9

  • Utilize inducible expression systems for temporal control

• Data integration approaches:

  • Correlate Phospho-HIST1H1E (S35) dynamics with transcriptional changes

  • Map phosphorylation patterns to chromatin state transitions

  • Integrate with other epigenetic modifications (DNA methylation, other histone marks)

How can I design experiments to investigate the cross-talk between HIST1H1E phosphorylation and other epigenetic modifications?

Investigating cross-talk between HIST1H1E phosphorylation and other epigenetic modifications requires sophisticated experimental designs:

• Sequential ChIP (Re-ChIP) approach:

  • First round: Immunoprecipitate with Phospho-HIST1H1E (S35) antibody

  • Second round: IP with antibodies against other histone modifications (H3K27me3, H3K4me3, etc.)

  • Compare enrichment patterns to single ChIP results

  • Include appropriate controls for each round of IP

• Proximity-based detection methods:

  • Proximity ligation assays (PLA) to detect co-occurrence of modifications

  • FRET-based approaches using labeled antibodies

  • Mass spectrometry of intact nucleosomes to identify co-occurring modifications

• Perturbation studies:

  • Selective inhibition of writers/erasers of specific modifications

  • Monitor consequent changes in HIST1H1E phosphorylation

  • Track reciprocal effects on other modifications when S35 phosphorylation is manipulated

• Genomic distribution analysis:

  • Generate genome-wide maps of Phospho-HIST1H1E (S35) and other modifications

  • Perform correlation analyses at different genomic features

  • Identify domains with synergistic or antagonistic modification patterns

• Functional readouts:

  • Measure transcriptional outcomes using reporter assays

  • Assess chromatin accessibility changes using ATAC-seq

  • Evaluate replication timing alterations using Repli-seq

What are the best practices for quantifying Phospho-HIST1H1E (S35) levels in experimental samples?

For accurate quantification of Phospho-HIST1H1E (S35) levels:

• Western blot quantification:

  • Always normalize phospho-signal to total HIST1H1E

  • Include standard curves using recombinant phosphorylated protein

  • Use infrared or chemiluminescent detection systems with verified linear range

  • Apply appropriate statistical tests for multiple comparisons

• Immunofluorescence quantification:

  • Collect images under identical acquisition parameters

  • Measure nuclear intensity in at least 100 cells per condition

  • Use automated segmentation to eliminate selection bias

  • Apply background subtraction using nuclear-free areas

  • Express results as phospho/total HIST1H1E ratio

• Flow cytometry approaches:

  • Fix cells with 4% paraformaldehyde followed by 90% methanol permeabilization

  • Perform dual staining for phospho and total HIST1H1E

  • Include isotype and phosphatase-treated controls

  • Gate on specific cell populations if analyzing heterogeneous samples

• ELISA-based quantification:

  • Generate standard curves with phosphopeptides

  • Ensure equal loading by quantifying total protein

  • Include spike-in controls to assess recovery efficiency

  • Validate results using orthogonal methods

What is the recommended protocol for performing ChIP-seq with Phospho-HIST1H1E (S35) antibody?

A comprehensive ChIP-seq protocol for Phospho-HIST1H1E (S35):

• Sample preparation:

  • Crosslink cells with 1% formaldehyde for 10 minutes

  • Consider dual crosslinking for linker histones (1.5mM EGS for 30 minutes before formaldehyde)

  • Quench with 125mM glycine

  • Isolate nuclei using hypotonic lysis

• Chromatin preparation:

  • Sonicate chromatin to 200-500bp fragments

  • Include phosphatase inhibitors in all buffers

  • Verify fragmentation by agarose gel electrophoresis

  • Pre-clear chromatin with protein A/G beads

• Immunoprecipitation:

  • Use 5μg Phospho-HIST1H1E (S35) antibody per 25μg chromatin

  • Include appropriate controls (IgG, input, total HIST1H1E)

  • Incubate overnight at 4°C with rotation

  • Wash with progressively stringent buffers

• Library preparation considerations:

  • Repair DNA ends and adapt for sequencing

  • Size select for fragments of 150-300bp

  • Use minimum PCR cycles to avoid amplification bias

  • Include spike-in controls for normalization

• Data analysis pipeline:

  • Align to reference genome using Bowtie2

  • Call peaks with MACS2 using broad peak settings

  • Compare to total HIST1H1E distribution

  • Perform differential binding analysis between conditions

How can I validate the specificity of Phospho-HIST1H1E (S35) antibody in my experimental system?

To validate Phospho-HIST1H1E (S35) antibody specificity:

• Genetic approaches:

  • Generate HIST1H1E knockout cells as negative controls

  • Create S35A mutant (non-phosphorylatable) and S35D/E (phospho-mimetic) constructs

  • Compare antibody reactivity across these genetic models

• Biochemical validations:

  • Perform peptide competition assays using phosphorylated and non-phosphorylated peptides

  • Treat samples with lambda phosphatase to remove phosphorylation

  • Use CDK inhibitors to reduce S35 phosphorylation in vivo

  • Perform immunodepletion experiments

• Orthogonal methods:

  • Compare results with mass spectrometry

  • Use Phos-tag gel electrophoresis to separate phosphorylated species

  • Validate with multiple antibodies targeting the same modification

  • Compare reactivity across cell cycle stages (phosphorylation increases during mitosis)

• Cross-reactivity assessments:

  • Test antibody against other H1 variants with similar phosphorylation sites

  • Perform immunoprecipitation followed by mass spectrometry

  • Evaluate specificity in tissues/cells with different H1 variant expression patterns

How can Phospho-HIST1H1E (S35) antibody be used to investigate epigenetic dysregulation in cancer?

Phospho-HIST1H1E (S35) antibody can provide valuable insights into cancer epigenetics:

• Comparative profiling approaches:

  • Compare phosphorylation patterns between tumor and adjacent normal tissues

  • Analyze phosphorylation across cancer progression stages

  • Correlate with clinical outcomes and treatment responses

• Mechanistic studies:

  • Investigate altered CDK activity effects on HIST1H1E phosphorylation

  • Map phosphorylation changes to oncogene activation or tumor suppressor loss

  • Identify cancer-specific interaction partners of phosphorylated HIST1H1E

• Functional consequences assessment:

  • Compare chromatin accessibility changes at cancer-relevant loci

  • Analyze transcriptional consequences using CRISPR-engineered phospho-mutants

  • Evaluate effects on DNA damage response and genomic stability

• Therapeutic implications:

  • Monitor HIST1H1E phosphorylation changes in response to kinase inhibitors

  • Correlate phosphorylation status with drug resistance mechanisms

  • Investigate combination strategies targeting both histone modifications and signaling pathways

• Biomarker development:

  • Evaluate prognostic value of Phospho-HIST1H1E (S35) in tissue microarrays

  • Develop quantitative immunohistochemistry protocols

  • Correlate with other established epigenetic biomarkers

What methodological approaches are recommended for studying Phospho-HIST1H1E (S35) dynamics in neurodegenerative disease models?

Studying Phospho-HIST1H1E (S35) in neurodegenerative contexts requires specialized approaches:

• Tissue-specific considerations:

  • Use fresh-frozen tissue sections for optimal phospho-epitope preservation

  • Compare phosphorylation patterns in affected vs. unaffected brain regions

  • Consider laser capture microdissection for cell-type-specific analysis

• Disease model approaches:

  • Compare transgenic models with human post-mortem samples

  • Evaluate changes in induced pluripotent stem cell (iPSC)-derived neurons

  • Utilize brain organoids for developmental aspects

• Technical adaptations:

  • Optimize fixation protocols to preserve phospho-epitopes in post-mortem tissue

  • Use signal amplification methods for detection in small samples

  • Implement multiplexed IHC/IF to correlate with disease markers

• Functional correlations:

  • Analyze relationship with DNA damage accumulation

  • Investigate heterochromatin maintenance

  • Assess impact on cellular senescence and aging pathways

• Intervention strategies:

  • Test effects of disease-modifying compounds on phosphorylation patterns

  • Evaluate restoration of normal phosphorylation by targeting relevant kinases/phosphatases

  • Consider combinatorial approaches affecting multiple epigenetic pathways