HIST1H1E (Ab-147) Antibody

What is HIST1H1E and what is its role in chromatin structure?

HIST1H1E (also known as H1.4, Histone H1b, Histone H1s-4, or H1F4) is a linker histone protein that plays a crucial role in chromatin architecture. It binds to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. HIST1H1E is necessary for the condensation of nucleosome chains into higher-order structured fibers and acts as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation . This protein is encoded by the Histone Gene Cluster 1 Member E gene, which is part of a family of epigenetic regulator genes. The proper functioning of HIST1H1E is essential for maintaining genomic stability and proper gene expression patterns in cells. Mutations in this gene have been associated with Rahman syndrome (OMIM #617537), a recently described intellectual disability syndrome .

What are the validated applications for HIST1H1E (Ab-147) antibody?

The HIST1H1E (Ab-147) antibody has been validated for use in several experimental techniques with specific applications in epigenetic and nuclear signaling research. The primary validated applications include Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry (IHC) . For IHC applications, the recommended dilution range is 1:10-1:100, though optimal dilutions should be determined experimentally for specific tissue types and fixation methods. The antibody is raised against a synthetic peptide derived from amino acids 140-151 of the human Histone H1.4 protein, making it specifically useful for detecting human HIST1H1E . While these are the manufacturer-validated applications, researchers have also adapted this antibody for related techniques such as western blotting in experimental contexts, particularly in studies related to chromatin organization and epigenetic regulation.

How should HIST1H1E (Ab-147) antibody be stored and handled for optimal performance?

Proper storage and handling of HIST1H1E (Ab-147) antibody is critical for maintaining its activity and specificity. The antibody is supplied in liquid form with a preservative (0.03% Proclin 300) and constituents including 50% Glycerol and 0.01M PBS at pH 7.4 . Upon receipt, the antibody should be stored at either -20°C or -80°C for long-term stability. Repeated freeze-thaw cycles should be strictly avoided as they can significantly degrade antibody performance and lead to increased background signal . For working solutions, small aliquots should be prepared to minimize freeze-thaw cycles. When handling the antibody, researchers should use sterile techniques and avoid contamination. Prior to each use, the antibody solution should be gently mixed by inversion or mild vortexing to ensure uniformity without causing protein denaturation or aggregation that could compromise binding specificity and experimental results.

What are the optimal conditions for using HIST1H1E (Ab-147) in immunohistochemistry?

When using HIST1H1E (Ab-147) antibody for immunohistochemistry (IHC), several parameters need to be optimized for successful detection. First, sample fixation is critical—paraformaldehyde (4%) is generally recommended for histone proteins, with fixation times of 10-15 minutes for cell preparations and 24-48 hours for tissue samples. Antigen retrieval is essential due to the nuclear localization of HIST1H1E; heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) at 95-100°C for 20 minutes typically yields good results . For blocking, a 5-10% solution of normal serum from the same species as the secondary antibody in PBS with 0.1-0.3% Triton X-100 is recommended to reduce background.

The optimal primary antibody dilution range for HIST1H1E (Ab-147) is 1:10-1:100 , though researchers should perform titration experiments for their specific samples. Incubation should be conducted at 4°C overnight or at room temperature for 1-2 hours. For detection, a compatible secondary antibody system (HRP-conjugated or fluorophore-labeled) should be selected based on the desired visualization method. When developing the signal, special attention should be paid to incubation times to avoid over-development which can mask specific staining patterns of the chromatin-associated protein.

How should Western blot protocols be optimized for HIST1H1E (Ab-147)?

For optimal detection of HIST1H1E using Western blot, several methodological considerations are important. Sample preparation should include specialized nuclear extraction protocols to efficiently isolate histone proteins. RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP40 or 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors) is effective for cell lysis when working with histone proteins . Cells should be incubated with the lysis buffer for approximately 30 minutes on ice to ensure complete extraction.

Histone proteins are relatively small (HIST1H1E is approximately 22 kDa), so 12-15% SDS-PAGE gels are recommended for optimal resolution. Protein transfer to membranes should be performed using PVDF membranes (such as Immobilon-P) with either wet transfer (25V overnight) or semi-dry transfer (15V for 30-45 minutes) protocols . Blocking should be performed with 5% non-fat milk in TBS-T for 1 hour at room temperature. The HIST1H1E (Ab-147) antibody should be diluted in blocking buffer and incubated overnight at 4°C. For detection, an appropriate HRP-conjugated secondary antibody should be used followed by enhanced chemiluminescence detection. Loading controls should include housekeeping proteins like tubulin to normalize expression levels .

What controls should be included when using HIST1H1E (Ab-147) antibody?

When conducting experiments with HIST1H1E (Ab-147) antibody, proper controls are essential for result validation and troubleshooting. Positive controls should include cell lines or tissues known to express HIST1H1E, such as human embryonic stem cells or proliferating cell lines. Negative controls should include samples where HIST1H1E expression is absent or significantly reduced, such as certain differentiated cell types or tissues with knockdown/knockout of HIST1H1E.

For antibody validation controls, include a primary antibody omission control (incubate samples with buffer only), an isotype control (using non-specific rabbit IgG at the same concentration), and a blocking peptide control (pre-incubate the antibody with excess immunizing peptide before application to the sample). For Western blot applications, recombinant HIST1H1E protein can serve as a positive control, while housekeeping proteins like tubulin should be used as loading controls .

In studies of HIST1H1E variants associated with Rahman syndrome, appropriate controls might include wild-type HIST1H1E expression alongside mutant versions. In cellular models, such as the mouse embryonic stem cell model described for studying Rahman syndrome, proper controls would include unmodified parent cell lines and cells expressing non-mutated human H1.4 tagged in the same manner as the mutant protein .

How can HIST1H1E (Ab-147) be utilized in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) using HIST1H1E (Ab-147) antibody requires careful optimization to study HIST1H1E binding patterns across the genome. Begin with crosslinking cells using 1% formaldehyde for 10 minutes at room temperature, followed by quenching with 125 mM glycine. After cell lysis using appropriate buffers (containing protease inhibitors), chromatin should be sheared to 200-500 bp fragments using sonication or enzymatic digestion. The optimal sonication conditions must be determined empirically for each cell type.

For immunoprecipitation, use 2-5 μg of HIST1H1E (Ab-147) antibody per reaction, incubating with chromatin overnight at 4°C. Protein A/G magnetic beads are recommended for capture, with incubation for 2-4 hours at 4°C. Following stringent washing steps, reverse crosslinking should be performed at 65°C overnight. After protein digestion with proteinase K, DNA purification, and quantification, the immunoprecipitated DNA can be analyzed by qPCR, sequencing, or array-based methods.

For ChIP-seq applications specifically, library preparation should follow standard protocols with appropriate adapters. Analysis should focus on broad peaks rather than narrow binding sites, as HIST1H1E functions as a linker histone with more distributed binding patterns compared to sequence-specific transcription factors. Integration with other histone modification datasets can provide insights into the relationship between HIST1H1E occupancy and chromatin states in both normal cells and disease models like Rahman syndrome .

How can HIST1H1E (Ab-147) be used to investigate chromatin dynamics in Rahman syndrome models?

HIST1H1E (Ab-147) antibody can be leveraged to study chromatin dynamics in Rahman syndrome models, providing insight into how mutations affect histone function and chromatin organization. In cellular models expressing human H1.4 variants, such as the mouse embryonic stem cell model described in the literature, the antibody can be used to compare wildtype and mutant HIST1H1E localization patterns via immunofluorescence microscopy . This approach allows researchers to determine if mutations alter nuclear distribution or chromatin binding properties of the protein.

For biochemical characterization, the antibody can be employed in chromatin fractionation studies to assess whether mutant HIST1H1E proteins show altered chromatin association compared to wildtype. Co-immunoprecipitation experiments using HIST1H1E (Ab-147) can identify changes in protein-protein interactions that might contribute to disease pathology. When working with tagged versions of H1.4 (PA-GFP and HA-tagged), the antibody can be used alongside tag-specific antibodies to validate expression and localization .

To investigate genome-wide changes, ChIP-seq with HIST1H1E (Ab-147) can be performed in cellular models to map binding sites and identify regions with altered occupancy due to mutations. This data can be integrated with transcriptome analysis (RNA-seq) to correlate changes in HIST1H1E binding with gene expression alterations. For mechanistic studies, HIST1H1E (Ab-147) can be used in fluorescence recovery after photobleaching (FRAP) experiments to assess how mutations affect the dynamic exchange rate of HIST1H1E proteins on chromatin, providing insight into the functional consequences of mutations associated with Rahman syndrome .

What approaches can be used to study HIST1H1E post-translational modifications?

Studying post-translational modifications (PTMs) of HIST1H1E requires specialized approaches to detect and quantify various modification types. For global PTM profiling, immunoprecipitation using HIST1H1E (Ab-147) followed by mass spectrometry analysis provides comprehensive identification of modifications including phosphorylation, methylation, acetylation, and ubiquitination. For this approach, optimization of sample preparation to minimize PTM loss during processing is critical.

For targeted analysis of specific modifications, a combination of techniques is recommended. Western blotting using HIST1H1E (Ab-147) for immunoprecipitation followed by modification-specific antibodies can detect relative levels of modifications across experimental conditions. Alternatively, reverse the approach by immunoprecipitating with modification-specific antibodies and detecting with HIST1H1E (Ab-147).

For cellular localization of modified HIST1H1E, proximity ligation assays (PLA) combining HIST1H1E (Ab-147) with modification-specific antibodies allow visualization of specific modified forms within cellular compartments. When studying dynamic changes in PTMs, pulse-chase experiments with metabolic labeling can be combined with HIST1H1E immunoprecipitation to track modification turnover rates.

In the context of Rahman syndrome research, comparing PTM patterns between wildtype and mutant HIST1H1E is particularly valuable. Since mutations occur in the C-terminal domain where many PTMs typically reside, alterations in modification patterns may contribute to disease pathology. The engineered cellular models expressing tagged H1.4 variants provide an excellent system for such comparative analyses .

What are common causes of non-specific binding when using HIST1H1E (Ab-147) and how can they be mitigated?

Non-specific binding when using HIST1H1E (Ab-147) antibody can arise from several sources. One common cause is insufficient blocking, which can be addressed by increasing blocking agent concentration (5-10% normal serum or BSA) and extending blocking time to 1-2 hours. Background may also result from excessive antibody concentration; performing careful titration experiments to determine optimal concentration is essential .

Cross-reactivity with other histone H1 variants can occur due to sequence homology. This can be minimized by using higher dilutions of primary antibody and more stringent washing protocols. In some cases, pre-absorption of the antibody with recombinant non-target histone variants can improve specificity.

High background in immunohistochemistry applications may stem from endogenous peroxidase activity, which should be quenched with hydrogen peroxide treatment before antibody application. Inadequate washing between steps is another common issue; increasing wash duration and volume can significantly improve signal-to-noise ratio.

For Western blot applications, non-specific bands may appear due to protein degradation or cross-reactivity. Using fresh samples with protease inhibitors and optimizing extraction conditions can reduce degradation products. PVDF membranes typically provide better signal-to-noise ratio than nitrocellulose for histone proteins.

When working with fluorescent detection systems, autofluorescence can be problematic. This can be reduced by treating samples with sodium borohydride or including a quenching step with Sudan Black B before antibody incubation.

How should conflicting HIST1H1E detection results between different experimental methods be resolved?

When faced with conflicting results between different detection methods using HIST1H1E (Ab-147) antibody, a systematic troubleshooting approach is necessary. First, validate the antibody's specificity in each experimental system separately using knockdown/knockout controls or blocking peptides to confirm specific binding. If antibody performance varies between applications, it may indicate context-dependent epitope accessibility.

For discrepancies between immunostaining and biochemical methods, consider fixation-induced epitope masking. Some fixatives may alter protein conformation, making the epitope inaccessible. Try alternative fixation methods or antigen retrieval protocols to determine if this resolves the discrepancy. If Western blot results conflict with immunofluorescence, evaluate whether denaturation affects epitope recognition, as HIST1H1E (Ab-147) targets a specific peptide sequence (amino acids 140-151) .

Technical factors may contribute to conflicting results. For instance, the chromatin condensation state can affect HIST1H1E detection, as can cell cycle phase since histone deposition varies throughout the cell cycle. Standardize cell synchronization protocols across experiments to minimize this variable. Sample preparation differences can also lead to discrepancies; nuclear extraction efficiency varies between protocols and may influence detection sensitivity.

To resolve conflicts between techniques, perform orthogonal validation using alternative detection methods or independent antibodies targeting different HIST1H1E epitopes. In the case of mutant HIST1H1E proteins, such as those associated with Rahman syndrome, tag-based detection systems can provide an antibody-independent method to verify results .

What considerations are important when analyzing HIST1H1E expression in different tissues and cell types?

Analyzing HIST1H1E expression across different tissues and cell types requires careful consideration of several biological and technical factors. HIST1H1E expression levels naturally vary between cell types, with higher expression typically observed in proliferating cells compared to terminally differentiated cells. Therefore, normalization to appropriate reference genes or proteins is critical for meaningful comparisons.

Tissue-specific chromatin structure can affect antibody accessibility and epitope availability. More compact heterochromatin regions may require more aggressive antigen retrieval methods for IHC or more stringent extraction conditions for biochemical analyses. Cell cycle phase significantly impacts histone expression and distribution; researchers should consider cell cycle synchronization or co-staining with cell cycle markers when comparing HIST1H1E levels between samples.

In tissues associated with Rahman syndrome, such as brain tissue where abnormalities like slender corpus callosum have been observed on MRI, special consideration should be given to developmental stage when analyzing HIST1H1E expression . The facial, skeletal, and neurological phenotypes associated with HIST1H1E mutations suggest tissue-specific roles that may be reflected in expression patterns or post-translational modifications.

When analyzing patient-derived samples, genetic background effects should be considered, as variants in other genes may compensate for or exacerbate HIST1H1E dysfunction. For quantitative analyses, digital image analysis with appropriate thresholding is recommended for immunohistochemistry, while normalized densitometry should be used for Western blot quantification. In all cases, biological replicates from multiple individuals are essential to account for person-to-person variation in histone expression levels.

How is HIST1H1E (Ab-147) being used to study the molecular mechanisms of Rahman syndrome?

HIST1H1E (Ab-147) antibody is playing a crucial role in elucidating the molecular mechanisms underlying Rahman syndrome. Researchers are utilizing this antibody to investigate how protein-truncating variants (PTVs) in HIST1H1E affect protein function and chromatin organization. Studies have shown that these mutations cluster to a 94-base pair region in the HIST1H1E carboxy terminal domain, resulting in a frameshift that produces mutant proteins sharing the same 38 amino acid carboxy terminal motif . The antibody enables researchers to compare the expression, localization, and chromatin binding properties of wildtype versus mutant HIST1H1E proteins.

In cellular models, such as the genetically engineered mouse embryonic stem cells that express human H1.4 variants, HIST1H1E (Ab-147) is being used alongside tagged versions of the protein to study the consequences of mutations . These models enable the investigation of how mutations affect nuclear organization, chromatin compaction, and gene expression patterns. The antibody facilitates immunofluorescence studies to visualize protein distribution, co-immunoprecipitation experiments to identify altered protein interactions, and ChIP assays to map genome-wide binding changes.

Researchers are also exploring how HIST1H1E mutations affect the protein's interaction with DNA and other chromatin components. The mutant proteins are predicted to have a significantly reduced net positive charge (−6 to 10.9) compared to the wildtype protein (charge of 41), which likely alters their DNA binding properties and chromatin condensation capabilities . These studies are providing insights into how epigenetic dysregulation contributes to the intellectual disability and distinctive facial features characteristic of Rahman syndrome, potentially opening avenues for therapeutic interventions targeting chromatin regulation.

What emerging techniques are enhancing our understanding of HIST1H1E function in chromatin biology?

Cutting-edge techniques are revolutionizing our understanding of HIST1H1E's role in chromatin biology, with HIST1H1E (Ab-147) antibody serving as an essential tool in many of these approaches. Live-cell imaging combined with photoactivatable GFP-tagged H1.4, as demonstrated in the mouse embryonic stem cell model, allows researchers to track HIST1H1E dynamics in real-time, revealing how the protein exchanges on chromatin and responds to cellular signals . This approach is particularly valuable for comparing the mobility and binding kinetics of wildtype versus mutant proteins associated with Rahman syndrome.

High-resolution chromatin mapping techniques, including CUT&RUN and CUT&Tag, offer improved sensitivity and specificity over traditional ChIP-seq. When performed with HIST1H1E (Ab-147), these methods provide finer-scale mapping of binding sites across the genome with lower background and cell number requirements. Integration of these datasets with other epigenomic profiles creates comprehensive maps of chromatin states and their relationship to HIST1H1E occupancy.

Proximity-based labeling approaches, such as APEX2 or BioID fused to HIST1H1E, enable unbiased identification of protein interactions in living cells. When coupled with mass spectrometry, these techniques reveal the HIST1H1E interactome and how it changes in disease states. Single-cell technologies are also being applied to study heterogeneity in HIST1H1E distribution and function across cell populations, particularly valuable in neurodevelopmental contexts relevant to Rahman syndrome .

Cryo-electron microscopy and cryo-electron tomography are providing unprecedented structural insights into how HIST1H1E influences higher-order chromatin organization. These approaches, combined with super-resolution microscopy using HIST1H1E (Ab-147) for immunolocalization, are revealing how linker histones shape the 3D genome and how mutations may disrupt this organization in disease states.

How do HIST1H1E variants correlate with specific clinical manifestations in Rahman syndrome?

Research using HIST1H1E (Ab-147) antibody is helping to establish genotype-phenotype correlations in Rahman syndrome by enabling detailed molecular characterization of patient-specific variants. Studies of 30 patients with HIST1H1E mutations have revealed that all disease-causing variants cluster in a specific 94-base pair region of the carboxy terminal domain and result in the same shift in reading frame . This consistency at the molecular level explains the recognizable syndrome phenotype despite different specific mutations.

The clinical manifestations of Rahman syndrome include intellectual disability (ID), typically of moderate severity, and a distinctive facial appearance characterized by a high frontal hairline, full lower cheeks in early childhood, and in later childhood and adulthood, a strikingly high frontal hairline, frontal bossing, and deep-set eyes . Additional features include hypothyroidism, abnormal dentition, behavioral issues, cryptorchidism in males, skeletal anomalies, and cardiac anomalies. Brain MRI frequently shows abnormalities, with a slender corpus callosum being a common finding .

Molecular studies using HIST1H1E (Ab-147) are investigating how these variants affect chromatin regulation in different tissues, potentially explaining the tissue-specific manifestations of the syndrome. For instance, the consistent facial features might result from dysregulated gene expression during craniofacial development due to altered chromatin structure. Similarly, the neurological phenotypes likely reflect HIST1H1E's role in neurodevelopment, where proper chromatin organization is crucial for establishing neural circuits.

The development of cellular and animal models, such as the mouse embryonic stem cell model expressing human H1.4 variants, provides valuable systems for investigating how specific mutations correlate with cellular phenotypes . These models, combined with patient-derived cells, are helping researchers understand the molecular basis of clinical variability and identify potential targets for therapeutic intervention.