HIST1H1E (Ab-35) Antibody

What is HIST1H1E and what is its primary function in cellular biology?

HIST1H1E encodes histone H1.4, a member of the histone H1 family that plays a crucial role in chromatin organization. The protein binds to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. Histones H1 are necessary for the condensation of nucleosome chains into higher-order structured fibers. HIST1H1E also functions as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation . This protein is part of the histone cluster located on chromosome 6p22.2 and is expressed in various human tissues, particularly in replicating cells .

What are the recommended applications for HIST1H1E (Ab-35) antibody?

HIST1H1E (Ab-35) antibody has been validated for multiple experimental applications:

The antibody has been specifically tested with human embryonic stem cell lines and HEK293T cells with successful detection of both endogenous and overexpressed H1.4 proteins .

How should the HIST1H1E (Ab-35) antibody be stored to maintain optimal activity?

For proper maintenance of antibody activity, store HIST1H1E (Ab-35) antibody at -20°C or -80°C upon receipt. The antibody is supplied in liquid form with a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4. It is crucial to avoid repeated freeze-thaw cycles as this can significantly compromise antibody performance . For laboratories conducting long-term studies, aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles and maintain consistent experimental results.

What experimental controls should be included when using HIST1H1E (Ab-35) antibody for immunostaining?

When designing immunostaining experiments with HIST1H1E (Ab-35) antibody, include the following controls:

• Positive control: Use cell lines with confirmed HIST1H1E expression such as HEK293T cells or human embryonic stem cell lines (e.g., H7 cells) .

• Negative control: Use cells where HIST1H1E expression is absent or significantly reduced, or include:

  • Secondary antibody-only control (omit primary antibody)

  • Isotype control (use rabbit IgG at the same concentration)

  • Blocking peptide competition assay (pre-incubate antibody with the immunizing peptide)

• Specificity validation: Transfect cells with HIST1H1E expression constructs (e.g., pFUW-Flag and Myc tagged-H1.4 plasmid) alongside control plasmid (e.g., pFUW-vector) to confirm antibody specificity.

Proper controls ensure that signals observed are specific to HIST1H1E rather than non-specific binding or background fluorescence.

What is the optimal method for detecting HIST1H1E in human embryonic stem cells using immunofluorescence?

Based on validated protocols, the following method yields strong and clear H1.4 signals in nucleoli of human embryonic stem cells :

• Cell preparation: Plate human embryonic stem cells (e.g., H7 line) on Matrigel pre-coated plates and allow growth for 3 days.

• Fixation: Fix cells with 4% formaldehyde/PBS solution for 15 minutes at room temperature.

• Washing: Wash three times with PBS to remove fixative completely.

• Permeabilization: Permeabilize cell membranes with 0.1% Triton X-100/PBS for 10-15 minutes.

• Blocking: Block non-specific binding with 3% BSA/PBS solution for 1 hour at room temperature.

• Primary antibody incubation: Dilute HIST1H1E (Ab-35) antibody 1:200 in blocking solution and incubate overnight at 4°C.

• Secondary antibody detection: Use Alexa Fluor 488-conjugated secondary antibody (1:500 dilution) for 1 hour at room temperature.

• Nuclear counterstaining: Counterstain with DAPI to visualize nuclei.

This protocol has been validated to produce strong and clear H1.4 signals specifically in the nucleolus .

How can non-specific binding be minimized when using HIST1H1E (Ab-35) antibody in Western blot applications?

To minimize non-specific binding in Western blot applications using HIST1H1E (Ab-35) antibody:

• Optimize blocking conditions: Use 5% skim milk in TBST as demonstrated in validated protocols . For persistent background, consider alternative blocking agents such as 3-5% BSA or commercial blocking buffers.

• Antibody dilution optimization: Start with the recommended 1:1000 dilution for WB , then adjust based on signal-to-noise ratio. If background is high, increase the dilution (e.g., 1:2000).

• Washing optimization: Perform at least 3-5 washes with TBST, increasing duration (5-10 minutes each) if background persists.

• Sample preparation: Extract proteins using appropriate lysis buffers (e.g., GST IP buffer: 25 mM Tris-HCl pH8.0, 150 mM NaCl, 100 mM KCl, 2 mM EDTA, 1% NP-40, with proteinase inhibitor cocktail) . Ensure complete denaturation of proteins.

• Gel concentration: Use 12% PAGE gels for optimal resolution of histone proteins .

Implementing these measures has been shown to produce clean Western blot results with recognition of both endogenous H1.4 proteins and ectopically overexpressed H1.4 proteins without non-specific signals .

What are potential causes for inconsistent staining patterns when using HIST1H1E (Ab-35) antibody in immunohistochemistry?

Inconsistent staining patterns in IHC can result from several factors:

• Fixation artifacts: Overfixation or underfixation can mask epitopes. The recommended fixation is 4% formaldehyde/PBS for 15 minutes for cell preparations . Tissue samples may require optimization of fixation time.

• Antigen retrieval inefficiency: HIST1H1E epitopes may be masked by fixation. Test different antigen retrieval methods (heat-induced in citrate buffer pH 6.0 or Tris-EDTA pH 9.0).

• Cell cycle variation: HIST1H1E expression and localization can vary through the cell cycle. Synchronize cells when possible or analyze cell cycle markers in parallel.

• Antibody concentration: The recommended dilution range for IHC is 1:20-1:200 . Titrate the antibody to determine optimal concentration for specific sample types.

• Detection system sensitivity: For low expression samples, consider signal amplification systems like tyramide signal amplification.

• Chromatin state: Accessibility of the HIST1H1E epitope may be affected by chromatin compaction state. Consider treatment with HDAC inhibitors to assess impact on staining patterns.

Compare staining patterns with known HIST1H1E localization (primarily nuclear with enrichment in nucleoli) to verify specificity .

How can HIST1H1E (Ab-35) antibody be utilized to study the role of histone H1.4 in chromatin remodeling and gene regulation?

HIST1H1E (Ab-35) antibody can be employed in several advanced techniques to investigate histone H1.4's role in chromatin remodeling:

• Chromatin Immunoprecipitation (ChIP): While not explicitly listed in the product applications, this polyclonal antibody can be adapted for ChIP assays to map genomic binding sites of H1.4. Start with a 1:100 dilution and 3-5 μg antibody per ChIP reaction. Cross-linking with 1% formaldehyde for 10 minutes is typically sufficient for histone studies.

• ChIP-seq: Combine ChIP with next-generation sequencing to generate genome-wide H1.4 binding profiles. This approach can reveal relationships between H1.4 occupancy and gene expression, especially in the context of differentiation or disease models.

• Co-immunoprecipitation (Co-IP): Use the antibody to pull down H1.4 and identify interacting proteins, providing insights into how H1.4 participates in chromatin remodeling complexes. The GST IP buffer described for protein extraction (25 mM Tris-HCl pH8.0, 150 mM NaCl, 100 mM KCl, 2 mM EDTA, 1% NP-40) can be adapted for Co-IP.

• Proximity ligation assay (PLA): Combine HIST1H1E (Ab-35) antibody with antibodies against suspected interaction partners to visualize and quantify protein-protein interactions in situ.

• FRAP (Fluorescence Recovery After Photobleaching): Use the antibody to create fluorescently tagged H1.4 constructs for studying the dynamics of H1.4 binding to chromatin in living cells.

These approaches can reveal how HIST1H1E/H1.4 contributes to gene regulation through chromatin structure modulation, particularly in developmental contexts or disease states.

How can HIST1H1E (Ab-35) antibody be used to investigate the role of H1.4 in disease pathogenesis, particularly in HIST1H1E-related syndromes?

HIST1H1E mutations have been implicated in a developmental disorder previously called "Rahman Syndrome," characterized by intellectual disability, dysmorphic features, and accelerated aging . To investigate H1.4's role in disease pathogenesis:

• Mutation-specific studies: Compare H1.4 localization using immunofluorescence with HIST1H1E (Ab-35) antibody in patient-derived cells versus controls. The validated protocol using 1:200 dilution for IF in fixed cells can be employed to detect differences in nuclear distribution patterns.

• Functional genomics approach:

  • Create isogenic cell lines with CRISPR-Cas9 to introduce pathogenic HIST1H1E frameshift mutations

  • Use HIST1H1E (Ab-35) antibody (1:1000 dilution for WB) to confirm expression of truncated proteins

  • Perform ChIP-seq to map altered binding profiles of mutant H1.4

• Cellular senescence investigation: As HIST1H1E mutations have been linked to accelerated senescence , use the antibody to study:

  • Co-localization with senescence markers

  • Chromatin changes during cellular aging

  • Altered protein interactions in senescent cells

• Methylation profiling: The aberrant function of the C-terminal tail of HIST1H1E has been associated with specific methylation profiles . Combine HIST1H1E (Ab-35) antibody ChIP with methylation analyses to correlate H1.4 binding with epigenetic changes in patient samples.

• Disease modeling: Apply the antibody in iPSC-derived neural models from patients with HIST1H1E mutations to investigate developmental abnormalities, using established immunostaining protocols .

This multifaceted approach can provide insights into how HIST1H1E mutations disrupt chromatin architecture and lead to the observed clinical phenotypes.

How does the performance of HIST1H1E (Ab-35) antibody compare with other commercially available HIST1H1E antibodies for studying histone modifications?

When comparing HIST1H1E (Ab-35) antibody to other commercially available options:

• Epitope specificity: HIST1H1E (Ab-35) antibody recognizes a sequence around Ser-35 of human histone H1.4 , while other antibodies may target different regions:

  • HIST1H1E (Ab-25) targets a region around amino acids 25-37

  • Some antibodies target post-translational modifications (e.g., acLys16)

• Cross-reactivity profile: HIST1H1E (Ab-35) has been validated specifically against human samples . Consider cross-reactivity needs when studying HIST1H1E across species.

• Application versatility: HIST1H1E (Ab-35) has been validated for ELISA and IF , while HIST1H1E (Ab-25) has demonstrated effectiveness in WB, IHC, and IF applications , making application requirements an important selection factor.

• Detection sensitivity: In validated experiments, HIST1H1E (Ab-25) successfully detected both endogenous and overexpressed H1.4 without non-specific signals in Western blot at 1:1000 dilution , providing a benchmark for comparison.

• Modification-specific detection: For studies focusing on specific post-translational modifications, specialized antibodies like anti-HIST1H1E acLys16 would be more appropriate than the sequence-specific HIST1H1E (Ab-35).

Select the antibody based on your specific experimental goals, required applications, and whether you need to detect total HIST1H1E or specific modified forms.

What methodological approaches can be used to distinguish between different histone H1 variants when studying chromatin structure?

Distinguishing between histone H1 variants requires specialized methodological approaches:

• Isoform-specific antibody selection:

  • Use HIST1H1E (Ab-35) antibody which specifically targets H1.4 (HIST1H1E) around Ser-35

  • Validate specificity through Western blot analysis comparing overexpressed tagged H1.4 with endogenous protein

  • Consider epitope location when designing experiments, as accessibility may differ in native chromatin

• Mass spectrometry-based approaches:

  • Combine immunoprecipitation using HIST1H1E (Ab-35) antibody with LC-MS/MS

  • Use targeted proteomics to quantify variant-specific peptides

  • Analyze post-translational modifications that distinguish variants

• ChIP-seq comparative analysis:

  • Perform parallel ChIP-seq with antibodies against different H1 variants

  • Compare genomic distribution patterns to identify variant-specific functions

  • Correlate with gene expression data to determine functional consequences

• Genetic manipulation strategies:

  • Use CRISPR-Cas9 to tag endogenous H1 variants with different epitopes

  • Create variant-specific knockouts to study compensation mechanisms

  • Employ directed mutagenesis of specific residues to analyze functional domains

• Microscopy-based differentiation:

  • Use super-resolution microscopy with HIST1H1E (Ab-35) antibody (1:50-1:200 dilution)

  • Employ fluorescence correlation spectroscopy to analyze binding dynamics

  • Study co-localization with other chromatin marks

These approaches can reveal variant-specific functions in chromatin organization and gene regulation that might be obscured when studying H1 histones as a group.

How might HIST1H1E (Ab-35) antibody be utilized in studying the role of H1.4 in cellular senescence and accelerated aging?

Recent research has established a link between HIST1H1E mutations, cellular senescence, and accelerated aging . HIST1H1E (Ab-35) antibody can be instrumental in exploring these connections:

• Senescence marker correlation studies:

  • Use immunofluorescence with HIST1H1E (Ab-35) antibody (1:50-1:200 dilution) to track H1.4 distribution changes during cellular senescence

  • Combine with markers like p16INK4a, p21, and SA-β-gal to correlate chromatin changes with senescence progression

  • Apply in models of premature aging syndromes to identify common pathways

• Chromatin accessibility analysis:

  • Combine HIST1H1E (Ab-35) antibody ChIP with ATAC-seq to correlate H1.4 binding with changes in chromatin accessibility during senescence

  • Investigate senescence-associated heterochromatin foci (SAHF) formation and H1.4 dynamics

  • Study the interplay between H1.4 and heterochromatin proteins in aging cells

• Longitudinal aging studies:

  • Monitor H1.4 levels and distribution in cellular models of aging using HIST1H1E (Ab-35) antibody Western blot (1:500-1:2000 dilution)

  • Investigate age-related post-translational modifications that might affect H1.4 function

  • Examine how H1.4 alterations affect tissue regeneration in aging models

• Therapeutic intervention assessment:

  • Use the antibody to evaluate chromatin changes after treatment with senolytic drugs

  • Monitor H1.4 dynamics in response to interventions that extend lifespan

  • Study the effects of epigenetic modulators on H1.4 distribution in senescent cells

This research could provide insights into how chromatin remodeling contributes to the aging process and identify potential therapeutic targets for age-related disorders.

What are the potential applications of HIST1H1E (Ab-35) antibody in single-cell epigenomic studies?

Emerging single-cell technologies offer new opportunities for applying HIST1H1E (Ab-35) antibody in epigenomic research:

• Single-cell CUT&Tag/CUT&RUN:

  • Adapt HIST1H1E (Ab-35) antibody protocols for targeted chromatin profiling at single-cell resolution

  • Start with 0.5-1 μg antibody per reaction, optimizing based on signal strength

  • Combine with transcriptome analysis to correlate H1.4 binding with gene expression heterogeneity

• Spatial epigenomics:

  • Apply HIST1H1E (Ab-35) antibody in spatial genomics platforms to map H1.4 distribution in tissue contexts

  • Use the validated IF protocol (1:50-1:200 dilution) as a starting point, optimizing for tissue preservation

  • Correlate H1.4 patterns with cellular identities and microenvironmental factors

• Multi-omics integration:

  • Combine single-cell HIST1H1E (Ab-35) ChIP-seq with single-cell RNA-seq and ATAC-seq

  • Develop computational frameworks to integrate multi-modal data

  • Identify cell-specific regulatory networks involving H1.4

• Live-cell dynamics:

  • Develop approaches to track H1.4 dynamics in living cells at single-molecule resolution

  • Study how individual cells respond to perturbations in real-time

  • Investigate cell-to-cell variability in H1.4 function during differentiation or stress response

• Disease heterogeneity analysis:

  • Apply in patient-derived samples to understand cellular heterogeneity in HIST1H1E-related disorders

  • Identify cell populations with distinct epigenetic signatures in disease states

  • Track epigenetic changes during disease progression at single-cell resolution

These applications represent the frontier of epigenetic research and could reveal unprecedented insights into how H1.4 contributes to cellular identity, differentiation, and disease pathogenesis.