HIST1H1E (Ab-51) Antibody

What is HIST1H1E and what biological functions does it serve?

HIST1H1E (also known as H1.4, H1E, or H1F4) is a linker histone protein that binds to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. This protein plays a crucial role in the condensation of nucleosome chains into higher-order structured fibers. Beyond its structural role, HIST1H1E functions as a regulator of individual gene transcription through multiple mechanisms including chromatin remodeling, nucleosome spacing, and DNA methylation . The protein is part of the H1 histone family and contributes significantly to genome organization and expression regulation.

What are the optimal storage conditions for HIST1H1E (Ab-51) antibody?

For maximum stability and performance, HIST1H1E (Ab-51) antibody should be stored at either -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can compromise antibody integrity and function. The antibody is typically supplied in liquid form with a buffer composition of 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . When planning experiments, it's advisable to prepare working aliquots to minimize freeze-thaw cycles of the stock solution.

What applications is the HIST1H1E (Ab-51) antibody validated for?

The HIST1H1E (Ab-51) antibody has been validated for ELISA (Enzyme-Linked Immunosorbent Assay) and IF (Immunofluorescence) applications. For immunofluorescence specifically, the recommended dilution range is 1:50-1:200 . When establishing a new experimental protocol, it is advisable to perform a titration experiment with multiple dilutions within this range to determine optimal antibody concentration for your specific cell type or tissue. The polyclonal nature of this antibody means it may detect multiple epitopes on the target protein, potentially increasing sensitivity but requiring careful validation for specificity.

How should I perform immunofluorescence experiments using HIST1H1E (Ab-51) antibody?

For immunofluorescence detection of HIST1H1E using this antibody:

• Culture cells on coverslips coated with either 0.01% poly-L-lysine or 0.2% gelatin, depending on your cell type

• Fix cells with 3% paraformaldehyde for 10 minutes at room temperature

• Permeabilize with 0.5% Triton X-100

• Block in a buffer containing 1% BSA, 22.52 mg/mL glycine in PBST (0.1% Tween 20 in PBS) for 30 minutes

• Incubate with HIST1H1E (Ab-51) primary antibody (diluted 1:50-1:200) for 1 hour at room temperature

• Wash three times with PBST

• Incubate with appropriate Alexa Fluor secondary antibody for 1 hour at room temperature

• Counterstain with DAPI to visualize nuclei

• Mount slides and image using fluorescent or confocal microscopy

This protocol has been successfully employed in studies examining nuclear localization patterns of histone proteins and can be adapted for co-localization studies with other chromatin components.

How can HIST1H1E (Ab-51) antibody be utilized in chromatin immunoprecipitation (ChIP) experiments?

While not explicitly listed among the validated applications, researchers experienced with HIST1H1E antibodies have adapted protocols for ChIP analysis. For optimal results:

• Crosslink chromatin using 1% formaldehyde for 10 minutes at room temperature

• Quench with 125 mM glycine for 5 minutes

• Lyse cells and sonicate chromatin to fragments of approximately 200-500 bp

• Pre-clear chromatin with protein A/G beads

• Immunoprecipitate with HIST1H1E (Ab-51) antibody (5-10 μg per reaction) overnight at 4°C

• Capture antibody-chromatin complexes using protein A/G beads

• Wash stringently to remove non-specific binding

• Reverse crosslinks and purify DNA for downstream analysis

For quantitative assessment, compare enrichment to input and IgG control. Expect enrichment at heterochromatic regions and repressed genes due to HIST1H1E's role in chromatin compaction. Since HIST1H1E belongs to a family of highly similar proteins, validation of ChIP specificity through knockout controls is strongly recommended.

How can I distinguish between normal and Rahman syndrome-associated HIST1H1E variants in research?

Rahman syndrome is associated with de novo frameshift mutations within the region coding for the C-terminal domain (CTD) of HIST1H1E. These mutations typically cluster within a 94-bp region in the CTD of the gene . To distinguish between normal and mutant variants:

Table 1: Comparison of Wild-type and Rahman Syndrome HIST1H1E Variants

For experimental detection, standard HIST1H1E antibodies may not distinguish between wild-type and mutant variants. Consider employing:

• Genotyping with primers flanking the mutation hotspot

• Western blotting to detect size differences (mutants typically show altered migration)

• Engineered tagged constructs (e.g., PA-GFP-H1.4) as implemented in cellular models

What methodological approaches can be used to study HIST1H1E dynamics in living cells?

To investigate the real-time dynamics of HIST1H1E in living cells, researchers have developed several sophisticated approaches:

• Photoactivatable GFP (PA-GFP) tagging: As demonstrated in the mouse embryonic stem cell model for Rahman syndrome, HIST1H1E can be tagged with PA-GFP . This approach allows:

  • Precise activation of fluorescence in specific nuclear regions

  • Measurement of protein mobility and exchange rates between chromatin-bound and free states

  • Comparison between wild-type and mutant protein dynamics

• Fluorescence Recovery After Photobleaching (FRAP):

  • Bleach a small region of interest containing fluorescently-tagged HIST1H1E

  • Record recovery of fluorescence over time

  • Calculate binding/unbinding kinetics from recovery curves

  • Compare residence times across different chromatin states

• Single-molecule tracking:

  • Label HIST1H1E with photoconvertible fluorophores

  • Track individual molecules over time

  • Analyze diffusion coefficients and binding events

  • Determine differences in mobility between euchromatin and heterochromatin regions

When implementing these approaches, careful controls must be performed to ensure that tagging does not disrupt native protein function or localization.

How can HIST1H1E (Ab-51) antibody be utilized in studying diseases associated with chromatin dysregulation?

HIST1H1E mutations have been implicated in Rahman syndrome, a neurodevelopmental disorder characterized by intellectual disability and overgrowth . To apply HIST1H1E (Ab-51) antibody in studying chromatin dysregulation:

• Comparative immunofluorescence:

  • Compare nuclear distribution patterns between patient-derived and control cells

  • Quantify signal intensity across different nuclear compartments

  • Co-stain with markers of heterochromatin (H3K9me3, HP1β) to assess chromatin organization

• Chromatin accessibility studies:

  • Combine antibody staining with ATAC-seq or DNase hypersensitivity assays

  • Correlate HIST1H1E localization with regions of altered chromatin accessibility

  • Investigate relationship between antibody binding sites and differential gene expression

• Protein interaction networks:

  • Use HIST1H1E (Ab-51) for co-immunoprecipitation followed by mass spectrometry

  • Identify differential protein interactions between wild-type and disease states

  • Map interaction changes to pathways involved in neurodevelopment

These approaches can provide insights into how HIST1H1E variants contribute to pathogenesis beyond simple descriptive observations.

What controls should be included when validating HIST1H1E (Ab-51) antibody specificity?

Comprehensive validation of HIST1H1E (Ab-51) antibody specificity requires multiple controls:

• Genetic controls:

  • HIST1H1E knockout or knockdown cells (negative control)

  • Cells overexpressing HIST1H1E (positive control)

  • Cells expressing tagged HIST1H1E for co-localization studies

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Observe elimination of specific signal in immunoassays

• Cross-reactivity assessment:

  • Test reactivity against related H1 variants (H1.1-H1.5)

  • Evaluate species cross-reactivity if working with non-human models

• Antibody dilution series:

  • Perform titration experiments to determine optimal concentration

  • Verify signal-to-noise ratio across dilution range

• Application-specific controls:

  • For IF: secondary antibody only control

  • For Western blotting: molecular weight verification and loading controls

  • For ChIP: IgG and input controls

Thorough validation is particularly important given the high sequence similarity between histone H1 family members.

What are the most common technical challenges when using HIST1H1E (Ab-51) antibody and how can they be addressed?

Table 2: Common Technical Challenges and Solutions

For particularly challenging applications, consider using alternative detection methods, such as genetically encoded tags (HA, FLAG) for which highly specific antibodies are available .

How can I optimize chromatin extraction protocols for maximum HIST1H1E detection?

Optimization of chromatin extraction for efficient HIST1H1E detection requires consideration of this protein's dynamic association with chromatin:

• Fixation optimization:

  • For chemical crosslinking: Use 1-2% formaldehyde for 10-15 minutes (avoid over-fixation)

  • For native chromatin: Extract in low-salt buffers (≤150 mM NaCl) to maintain H1 association

• Extraction buffer composition:

  • Include divalent cations (2-5 mM MgCl₂) to stabilize chromatin structure

  • Add protease inhibitors to prevent HIST1H1E degradation

  • Consider phosphatase inhibitors to maintain native phosphorylation state

• Fractionation approach:

  • For comprehensive HIST1H1E analysis, collect multiple chromatin fractions using sequential extraction:
    a. Low salt buffer (e.g., 10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl₂, 0.5% NP-40) for soluble fraction
    b. Nuclease digestion (MNase) for loosely bound chromatin fraction
    c. High salt extraction (300-600 mM NaCl) for tightly bound fraction

• Handling considerations:

  • Perform extractions at 4°C to minimize protein degradation

  • Avoid excessive mechanical shearing before intended fragmentation

  • For ChIP applications, optimize sonication to achieve 200-500 bp fragments

These optimizations enhance HIST1H1E retention and detection across multiple experimental platforms.

How can I establish a cellular model to study HIST1H1E mutations related to Rahman syndrome?

Based on recent research, a sophisticated cellular model for Rahman syndrome can be established using CRISPR/Cas9 genome engineering in murine embryonic stem cells (mESCs) . The key methodological steps include:

• Design and cloning of CRISPR components:

  • Design sgRNAs targeting the mouse H1.4 locus using tools like Chopchop

  • Clone sgRNAs into a Cas9-GFP expression vector (e.g., pX458)

  • Create a targeting vector containing:

    • 5' and 3' homology arms from mouse H1.4 locus

    • Human H1.4 gene with HA tag

    • Promoterless neomycin selection marker flanked by loxP sites

    • Photoactivatable GFP (PA-GFP) tag

• Cell transfection and selection:

  • Transfect mESCs with the CRISPR/Cas9 and targeting vectors

  • Select transfected cells with Geneticin (200 μg/mL) for 2 weeks

  • Pick and expand drug-resistant colonies with undifferentiated morphology

• Genotyping and validation:

  • Extract DNA from selected clones

  • Perform PCR to confirm successful recombination at both 5' and 3' homology arms

  • Use restriction enzyme digestion (e.g., HindIII) to determine zygosity

  • Validate expression through RT-PCR and western blotting

• Cre recombinase-mediated induction:

  • Treat validated clones with Cre Recombinase Adenovirus

  • Allow 36 hours for transgene expression

  • Confirm induction through RT-PCR and western blotting

This model enables detailed investigations of HIST1H1E dynamics, including protein interactions, nuclear localization, and functional consequences of Rahman syndrome mutations .

What cell-based assays can be performed to analyze functional consequences of HIST1H1E mutations?

Several cell-based assays can provide insights into the functional impact of HIST1H1E mutations:

• Chromatin relaxation assessment:

  • MNase digestion assay to measure chromatin accessibility

  • ATAC-seq to map open chromatin regions genome-wide

  • DNase-seq to identify hypersensitive sites

• Histone modification analysis:

  • Immunofluorescence or ChIP for key histone marks (H3K4me2, H3K9me3, H3K27me3)

  • Western blotting to quantify global levels of histone modifications

  • Mass spectrometry for comprehensive PTM profiling

• Cell proliferation and cycle analysis:

  • Growth curve generation by daily cell counting

  • Flow cytometry with propidium iodide staining for cell cycle distribution

  • EdU incorporation assays for DNA synthesis assessment

• Gene expression profiling:

  • RNA-seq to identify differentially expressed genes

  • RT-qPCR validation of key target genes

  • Analysis of pluripotency markers (Sox2, Oct4) in stem cell models

• Protein interaction studies:

  • Co-immunoprecipitation with HA-tagged HIST1H1E

  • Proximity ligation assay for in situ detection of protein interactions

  • ChIP-seq to map genome-wide binding patterns

These assays have revealed that Rahman syndrome mutations lead to enhanced chromatin relaxation, reduced methylation of specific histone H3 lysine residues, and decreased binding of heterochromatin protein HP1β .

How do post-translational modifications affect HIST1H1E function and antibody recognition?

Post-translational modifications (PTMs) of HIST1H1E play critical roles in regulating its function and can impact antibody recognition:

Table 3: Major HIST1H1E Post-Translational Modifications and Their Effects

When using HIST1H1E (Ab-51) antibody, which targets the region around Lys-51, consider that modifications at or near this residue could affect antibody binding efficiency. If studying specific PTMs, specialized phospho-specific or other modification-specific antibodies may be required. For comprehensive PTM analysis, mass spectrometry approaches are recommended to complement antibody-based detection methods.

How does HIST1H1E contribute to higher-order chromatin organization?

Recent structural studies have provided significant insights into HIST1H1E's role in chromatin architecture:

• Nucleosome binding asymmetry:

  • The HIST1H1E globular domain contacts the nucleosome dyad and both DNA linkers

  • The C-terminal domain (CTD) interacts mainly with a single linker

  • This asymmetric binding induces directional compaction of chromatin fibers

• Linker DNA organization:

  • HIST1H1E draws the two linker DNA segments together

  • It reduces linker DNA flexibility and dynamics

  • The nucleosome dyad likely determines CTD collapse on distinct DNA arms

• Chromatin fiber formation:

  • By bringing linker DNA segments closer, HIST1H1E promotes formation of zigzag-type chromatin fibers

  • HIST1H1E stabilizes nucleosome-nucleosome contacts in higher-order structures

  • The CTD's high positive charge neutralizes negative charges on linker DNA

These structural insights explain why Rahman syndrome mutations in the CTD significantly impact chromatin organization, leading to enhanced chromatin relaxation and altered gene expression patterns observed in patient cells .

What are promising future research directions utilizing HIST1H1E (Ab-51) antibody?

Several promising research directions could benefit from application of HIST1H1E (Ab-51) antibody:

• Single-cell epigenomics:

  • Combining antibody-based detection with single-cell technologies

  • Mapping HIST1H1E distribution in heterogeneous cell populations

  • Correlating HIST1H1E binding patterns with cell-specific gene expression programs

• Brain development and neurological disorders:

  • Investigating HIST1H1E's role in neural differentiation

  • Comparing chromatin organization in control versus Rahman syndrome models

  • Exploring potential therapeutic approaches targeting chromatin structure

• Epigenetic inheritance:

  • Examining HIST1H1E dynamics during gametogenesis and early embryonic development

  • Assessing potential transgenerational effects of altered HIST1H1E function

  • Investigating interactions with DNA methylation machinery

• Chromatin phase separation:

  • Exploring HIST1H1E's contribution to biomolecular condensate formation

  • Analyzing how CTD mutations affect phase separation properties

  • Developing assays to visualize and quantify chromatin compartmentalization

• Therapeutic targeting:

  • Screening for compounds that normalize chromatin structure in disease models

  • Developing peptide-based approaches to mimic normal HIST1H1E function

  • Exploring gene therapy approaches for Rahman syndrome

These research directions could significantly advance our understanding of chromatin biology and potentially lead to therapeutic interventions for chromatin-related disorders.