HIST1H1E (Ab-62) Antibody

What are the key specifications of HIST1H1E (Ab-62) Antibody?

HIST1H1E (Ab-62) Antibody is a rabbit-derived polyclonal IgG antibody that specifically targets human Histone H1.4 protein, particularly around the Lysine-62 site. This unconjugated antibody has been validated for multiple research applications including ELISA, Western Blotting (WB), and Immunohistochemistry (IHC) . The antibody's immunogen is a peptide sequence around the site of Lysine-62 derived from Human Histone H1.4, making it highly specific for this target .

The antibody targets a protein also known by several synonyms including H1 histone family member 4, H1.4, H14_HUMAN, H1E, H1F4, Histone 1 H1e, Histone cluster 1 H1e, and others . When considering storage conditions, the antibody should be aliquoted and stored at -20°C, with repeated freeze/thaw cycles avoided to maintain reactivity .

What biological functions does the target protein HIST1H1E/H1.4 serve?

HIST1H1E encodes Histone H1.4, a member of the somatic, replication-dependent linker histone subfamily that plays critical roles in chromatin organization and gene regulation. As a linker histone, H1.4 binds to nucleosomes at DNA entry and exit sites, facilitating higher-order chromatin structure formation and compaction .

Recent research has revealed that H1.4 has multiple functions:

• Chromatin compaction and organization

• Regulation of gene expression through modulation of DNA accessibility

• Involvement in cellular senescence pathways

• Contribution to development processes

Notably, mutations in the C-terminal tail of HIST1H1E have been causally linked to a syndrome that includes intellectual disability (MIM: 617537) and premature aging phenotypes . These mutations appear to disrupt proper chromatin compaction, affecting cellular proliferation and accelerating cellular senescence.

What are the recommended applications and dilutions for HIST1H1E (Ab-62) Antibody?

HIST1H1E (Ab-62) Antibody has been validated for several key research applications:

Researchers should note that optimal dilutions/concentrations should be determined empirically for each specific experimental setup, as factors such as sample type, preparation method, and detection system can influence performance . The antibody has been purified using antigen affinity chromatography, which enhances its specificity for the target epitope .

How should I design ChIP experiments using HIST1H1E (Ab-62) Antibody?

When designing Chromatin Immunoprecipitation (ChIP) experiments with HIST1H1E (Ab-62) Antibody, follow these methodological considerations:

• Experimental controls:

  • Include input chromatin as a reference sample (5-10% of starting material)

  • Use anti-H3 antibody as a positive control for general chromatin abundance

  • Include isotype-matched IgG as a negative control to assess non-specific binding

• Sample preparation:

  • Crosslinking: Optimize formaldehyde concentration (typically 1%) and time (8-10 minutes)

  • Sonication: Adjust conditions to achieve chromatin fragments of 200-500 bp

  • Pre-clearing: Use protein A/G beads to reduce background

• Immunoprecipitation:

  • Antibody concentration: Start with 2-5 μg per reaction

  • Incubation time: Overnight at 4°C with rotation

  • Washing: Use stringent wash buffers to reduce non-specific binding

• Analysis methods:

  • For ChIP-qPCR: Design primers for known targets and negative regions

  • For ChIP-seq: Include appropriate sequencing controls and bioinformatic analysis

  • For data normalization: Use input samples and normalize to total H3 occupancy

• Validation approach:

  • Verify enrichment at known H1.4-bound regions

  • Compare with published H1 variant distribution data

  • Consider multiplexing with other histone marks to understand chromatin context

This comprehensive approach ensures reliable data generation from ChIP experiments using HIST1H1E (Ab-62) Antibody .

What are the critical steps for optimizing Western blotting with HIST1H1E (Ab-62) Antibody?

Optimizing Western blotting with HIST1H1E (Ab-62) Antibody requires attention to several critical parameters:

• Sample preparation:

  • Nuclear extraction: Use specialized protocols to efficiently extract nuclear proteins

  • Histone enrichment: Consider acid extraction methods for histone purification

  • Protein quantification: Ensure equal loading (10-20 μg of nuclear extract)

  • Denaturation: Heat samples at 95°C for 5 minutes in reducing sample buffer

• Electrophoresis considerations:

  • Gel percentage: Use 15-18% SDS-PAGE for optimal histone resolution

  • Running conditions: 100-120V constant voltage

  • Molecular weight marker: Include to verify ~22 kDa band for H1.4

• Transfer optimization:

  • Membrane selection: PVDF membranes with 0.2 μm pore size work best for histones

  • Transfer method: Semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight

  • Transfer validation: Use reversible staining (Ponceau S) to confirm protein transfer

• Antibody incubation:

  • Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody: Start with 1:500 dilution (from the recommended 1:200-1:2000 range)

  • Incubation time: Overnight at 4°C with gentle rocking

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000 dilution

• Detection optimization:

  • Signal development: Enhanced chemiluminescence (ECL) substrate

  • Exposure time: Start with 30 seconds, then adjust as needed

  • Stripping and reprobing: Consider for multiple target analysis

Each of these parameters should be systematically optimized for your specific experimental system to achieve optimal signal-to-noise ratio.

How can I validate the specificity of HIST1H1E (Ab-62) Antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable data. For HIST1H1E (Ab-62) Antibody, implement a multi-faceted validation approach:

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (derived from Lys-62 region)

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be significantly reduced or eliminated in blocked samples

• Genetic validation:

  • Test antibody reactivity in cells with HIST1H1E knockdown or knockout

  • Compare with wild-type cells expressing normal levels of H1.4

  • The specific signal should be reduced proportionally to knockdown efficiency

• Recombinant protein validation:

  • Test antibody against purified recombinant H1.4 protein

  • Include other H1 variants to assess cross-reactivity

  • Evaluate dose-dependent detection

• Tagged protein approach:

  • Use cells expressing HA-tagged or GFP-tagged H1.4

  • Perform parallel detection with anti-tag antibodies and HIST1H1E (Ab-62)

  • Co-localization indicates specificity

• Mass spectrometry validation:

  • Perform immunoprecipitation with HIST1H1E (Ab-62) Antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm predominant enrichment of H1.4/HIST1H1E

This comprehensive validation strategy ensures that experimental results reflect true H1.4 biology rather than artifacts of non-specific binding.

How can HIST1H1E (Ab-62) Antibody be applied to study chromatin remodeling in cellular senescence?

Recent research has established a link between mutations in HIST1H1E, aberrant chromatin remodeling, and accelerated cellular senescence . HIST1H1E (Ab-62) Antibody can be instrumental in studying these connections through several sophisticated approaches:

• Chromatin accessibility analysis:

  • Perform ChIP-seq with HIST1H1E (Ab-62) Antibody in wild-type and mutant cells

  • Integrate with ATAC-seq or DNase-seq data to correlate H1.4 binding with chromatin accessibility

  • Focus analysis on senescence-associated genes and regulatory elements

  • Compare cells at different passages to track temporal changes during senescence progression

• Protein interaction network studies:

  • Use the antibody for co-immunoprecipitation experiments

  • Compare interacting partners between wild-type and mutant H1.4

  • Look for differential binding to chromatin remodeling complexes

  • Identify altered interactions that might explain senescence phenotypes

• Cellular senescence marker correlation:

  • Perform dual immunofluorescence with HIST1H1E (Ab-62) Antibody and senescence markers (p16, p21, γH2AX)

  • Quantify nuclear distribution patterns in relation to senescence status

  • Track temporal changes in H1.4 distribution during senescence induction

• Heterochromatin analysis:

  • Examine co-localization with heterochromatin markers (HP1α, H3K9me3)

  • Assess formation of senescence-associated heterochromatin foci (SAHF)

  • Quantify changes in heterochromatin distribution in cells with mutant H1.4

These approaches can provide mechanistic insights into how aberrant function of the C-terminal tail of HIST1H1E accelerates cellular senescence and causes premature aging phenotypes .

What considerations are important when using HIST1H1E (Ab-62) Antibody in developmental studies?

When studying developmental processes with HIST1H1E (Ab-62) Antibody, researchers should consider several specialized approaches:

• Cellular model systems:

  • Embryonic stem cell models provide excellent systems for studying H1.4 during differentiation

  • Consider using mouse ES cells with humanized HIST1H1E as described in recent studies

  • Patient-derived iPSCs can model developmental impacts of HIST1H1E mutations

• Developmental time-course analysis:

  • Track H1.4 expression and localization during differentiation processes

  • Compare with other H1 variants to identify stage-specific patterns

  • Correlate with expression of developmental markers and transcription factors

• Chromatin state mapping:

  • Perform ChIP-seq at key developmental transitions

  • Focus analysis on developmentally regulated genomic regions

  • Integrate with histone modification data to understand chromatin context

• Technical considerations:

  • Optimization for different tissue types and developmental stages

  • Fixation protocols may require adjustment for embryonic tissues

  • Signal normalization strategies to account for changing nuclear architecture

• Disease modeling approaches:

  • Implement CRISPR/Cas9 genome engineering to study specific mutations

  • Use conditional expression systems to control timing of mutant protein expression

  • Track phenotypic consequences of H1.4 dysfunction during development

These approaches can help elucidate the role of H1.4 in normal development and developmental disorders associated with HIST1H1E mutations, such as Rahman syndrome .

How can HIST1H1E (Ab-62) Antibody be utilized in lymphoma research?

Recent studies have identified mutations in linker histone H1 genes, including HIST1H1E, as drivers of peripheral lymphoid malignancies . HIST1H1E (Ab-62) Antibody can be applied to lymphoma research through these methodological approaches:

• Mutation-specific analysis:

  • Compare H1.4 distribution patterns between wild-type and mutant lymphoma samples

  • Correlate with lymphoma subtypes and clinical outcomes

  • Investigate how mutations affect H1.4 interaction with chromatin

• Epigenetic landscape characterization:

  • Perform ChIP-seq in lymphoma samples to map H1.4 distribution

  • Integrate with histone modification data (H3K27me3, H3K27ac)

  • Analyze changes at oncogene and tumor suppressor loci

• Chromatin accessibility studies:

  • Correlate H1.4 binding with chromatin accessibility changes in lymphoma

  • Focus on regulatory regions of genes involved in lymphocyte differentiation

  • Compare patterns across different lymphoma subtypes

• Therapeutic response monitoring:

  • Track H1.4 distribution changes following treatment with epigenetic modifiers

  • Correlate changes with treatment response

  • Identify potential biomarkers for therapy selection

• Technical considerations for lymphoid tissues:

  • Optimize fixation and extraction protocols specifically for lymphoid samples

  • Consider flow cytometry applications with permeabilization for rare subpopulation analysis

  • Develop multiplexed protocols including lymphoma classification markers

These approaches can provide insights into how alterations in H1.4 function contribute to lymphoma pathogenesis and potentially identify new therapeutic targets .

What are common issues when using HIST1H1E (Ab-62) Antibody and how can they be resolved?

When working with HIST1H1E (Ab-62) Antibody, researchers may encounter several technical challenges. Here are common issues and evidence-based solutions:

Systematic implementation of these solutions will help resolve common technical issues when working with HIST1H1E (Ab-62) Antibody.

How should researchers interpret changes in H1.4 localization patterns?

Interpreting changes in H1.4 localization detected with HIST1H1E (Ab-62) Antibody requires careful analysis and consideration of biological context:

This analytical framework helps convert observations of H1.4 localization into meaningful biological insights about chromatin organization and cellular state.

How can HIST1H1E (Ab-62) Antibody be integrated with cutting-edge genomic technologies?

Integration of HIST1H1E (Ab-62) Antibody with emerging genomic technologies opens new research possibilities:

• CUT&RUN and CUT&Tag applications:

  • Adapt the antibody for these higher-resolution chromatin profiling methods

  • Advantages include lower cell input requirements (1,000-50,000 cells)

  • Higher signal-to-noise ratio compared to traditional ChIP-seq

  • Enables profiling of rare cell populations or limited clinical samples

• Single-cell chromatin analysis:

  • Optimize protocols for single-cell CUT&Tag

  • Correlate H1.4 binding with chromatin accessibility at single-cell resolution

  • Identify cell subpopulations with distinct H1.4 distribution patterns

  • Track heterogeneity in disease progression

• Multi-omics integration:

  • Correlate H1.4 ChIP-seq data with:

    • RNA-seq to link H1.4 binding with gene expression

    • DNA methylation data to study epigenetic crosstalk

    • Hi-C data to explore H1.4's role in 3D genome organization

• Live-cell imaging approaches:

  • Combine with GFP-tagged H1.4 systems for validation

  • Study dynamics of H1.4 chromatin binding in real-time

  • Measure residence time on chromatin using FRAP

  • Track redistribution during differentiation or senescence

• Spatial genomics integration:

  • Combine immunofluorescence with in situ sequencing

  • Map H1.4 distribution in relation to genomic regions in intact cells/tissues

  • Preserve spatial context while gathering molecular information

These integrative approaches extend the utility of HIST1H1E (Ab-62) Antibody beyond traditional applications, enabling more comprehensive understanding of H1.4 function.

What novel methodological approaches can enhance HIST1H1E (Ab-62) Antibody applications in disease modeling?

Advanced methodological approaches using HIST1H1E (Ab-62) Antibody can significantly enhance disease modeling studies:

• CRISPR-engineered cellular models:

  • Generate HIST1H1E mutations in relevant cell types using CRISPR/Cas9

  • Create conditional expression systems for temporal control

  • Develop isogenic cell lines differing only in HIST1H1E status

  • Apply the antibody to track mutant protein behavior

• Patient-derived models:

  • Study H1.4 in patient-derived cells (fibroblasts, lymphocytes, iPSCs)

  • Compare with age-matched controls

  • Track disease progression through H1.4 distribution changes

  • Correlate with senescence markers in Rahman syndrome

• Therapeutic screening applications:

  • Use the antibody to monitor H1.4 distribution in drug screening assays

  • Target chromatin modifiers that might rescue aberrant H1.4 function

  • Develop high-content screening approaches incorporating H1.4 localization

• Chromatin conformation capture integration:

  • Combine with Hi-C or Micro-C to study 3D genome organization

  • Analyze topologically associating domain (TAD) boundaries in relation to H1.4 binding

  • Assess how HIST1H1E mutations affect chromatin loops and domains

• Mass spectrometry-based approaches:

  • Use HIST1H1E (Ab-62) Antibody for immunoprecipitation followed by mass spectrometry

  • Identify post-translational modifications on H1.4

  • Compare modification patterns between normal and disease states

  • Map the interactome of wild-type versus mutant H1.4

These methodological innovations can provide deeper insights into disease mechanisms associated with HIST1H1E mutations and potentially identify therapeutic targets for conditions like Rahman syndrome and lymphoid malignancies .