HIST1H1E (Ab-145) Antibody

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

HIST1H1E encodes Histone H1.4, a member of the linker histone H1 family. This protein binds to linker DNA between nucleosomes, helping to form the macromolecular structure known as the chromatin fiber . Histones H1, including HIST1H1E, are necessary for the condensation of nucleosome chains into higher-order structured fibers and regulate gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation .

The HIST1H1E gene is intronless and located within the large histone gene cluster on chromosome 6 . Proper function of HIST1H1E is critical for normal cellular processes, with mutations leading to HIST1H1E syndrome (also known as Rahman syndrome), characterized by intellectual disability and developmental abnormalities .

How does the HIST1H1E (Ab-145) antibody recognize its target protein?

The HIST1H1E (Ab-145) antibody specifically recognizes the region around Threonine 145 in human Histone H1.4 protein . This antibody was generated using a "Peptide sequence around site of Thr (145) derived from Human Histone H1.4" as the immunogen . It is a polyclonal antibody raised in rabbits and has been antigen affinity purified to enhance specificity . The antibody's specificity for this particular region makes it useful for detecting HIST1H1E in various experimental applications.

What are the common applications for HIST1H1E (Ab-145) antibody?

The HIST1H1E (Ab-145) antibody has been validated for several key research applications:

The antibody has been specifically validated for ChIP experiments using HeLa cells treated with Micrococcal Nuclease, where immunoprecipitated DNA was quantified using real-time PCR with primers against the β-Globin promoter .

What is the specificity of the HIST1H1E (Ab-145) antibody?

Based on the product information, the HIST1H1E (Ab-145) antibody is specific for human Histone H1.4 (HIST1H1E) . The antibody's species reactivity is limited to human samples, unlike some other HIST1H1E antibodies that may cross-react with mouse or rat samples . This human specificity makes it particularly valuable for studying human cell lines and tissues, but researchers should be aware of this limitation when working with non-human models.

How should HIST1H1E (Ab-145) antibody be stored and handled?

For optimal performance, the HIST1H1E (Ab-145) antibody should be stored according to these guidelines:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles

• The antibody is provided in liquid form in a storage buffer containing:

  • 0.03% Proclin 300 (preservative)

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

When handling the antibody for experiments, keep it on ice and avoid prolonged exposure to room temperature to preserve its activity.

How can HIST1H1E (Ab-145) antibody be used in ChIP experiments?

The HIST1H1E (Ab-145) antibody has been specifically validated for Chromatin Immunoprecipitation (ChIP) applications . Based on the available data, an optimized ChIP protocol using this antibody includes:

• Cross-link protein-DNA complexes using formaldehyde

• Lyse cells and isolate chromatin

• Fragment chromatin using sonication or enzymatic digestion with Micrococcal Nuclease

• Immunoprecipitate protein-DNA complexes using 5μg of HIST1H1E (Ab-145) antibody

• Include appropriate controls (such as normal rabbit IgG)

• Reverse cross-links and purify DNA

• Analyze precipitated DNA using qPCR, sequencing, or other methods

The validated ChIP experiment demonstrated in the product information used HeLa cells (4×10^6) treated with Micrococcal Nuclease, followed by sonication and immunoprecipitation with 5μg of the anti-HIST1H1E antibody . This approach allows researchers to study genomic localization of HIST1H1E and its association with specific regulatory regions.

How can HIST1H1E (Ab-145) antibody be used to study chromatin dynamics?

For investigating chromatin dynamics using HIST1H1E (Ab-145) antibody, researchers can implement several approaches:

• Cell Cycle Analysis: Since HIST1H1E expression varies throughout the cell cycle (primarily expressed in S phase) , researchers can synchronize cells and examine HIST1H1E localization and abundance at different cell cycle stages.

• Chromatin Compaction Studies: The antibody can be used to assess HIST1H1E distribution following treatments that affect chromatin structure, such as:

  • Histone deacetylase inhibitors

  • DNA methyltransferase inhibitors

  • Transcription activators or repressors

• CSK Buffer Treatment: As demonstrated in result , researchers can use CSK (cytoskeletal) buffer treatment prior to fixation to assess the chromatin binding stability of HIST1H1E.

• Co-localization Studies: Combining HIST1H1E (Ab-145) antibody with antibodies against other chromatin proteins can reveal dynamic interactions during cellular processes.

How does phosphorylation at Thr145 affect HIST1H1E function?

While the search results don't specifically address how phosphorylation at Thr145 affects biological function, we can infer several important aspects:

The C-terminal domain of HIST1H1E contains multiple serine/threonine residues that undergo reversible phosphorylation, which modulates the dynamics of chromatin compaction . Partial phosphorylation allows chromatin relaxation during interphase, while full phosphorylation is required for maximal chromatin condensation during mitosis .

Since the HIST1H1E (Ab-145) antibody targets the region around Threonine 145, it's important to consider how phosphorylation might affect antibody recognition. Researchers studying phosphorylation-dependent functions should determine whether this antibody recognizes the phosphorylated or non-phosphorylated form, as this will significantly impact experimental interpretation.

How can HIST1H1E (Ab-145) antibody be used to study Rahman syndrome?

Rahman syndrome (also known as HIST1H1E syndrome) is caused by frameshift mutations in the C-terminal domain of HIST1H1E . The HIST1H1E (Ab-145) antibody can be utilized in several approaches to study this rare disorder:

• Protein Expression Analysis: Western blotting can compare expression levels between wild-type and mutant HIST1H1E in patient-derived cells or engineered cellular models.

• Chromatin Binding Assessment: ChIP experiments can reveal how disease-causing mutations affect the genomic binding patterns of HIST1H1E .

• Cellular Senescence Investigation: HIST1H1E mutations are associated with accelerated cellular senescence and premature aging . The antibody can help study connections between mutant protein and senescence markers.

• DNA Methylation Studies: Mutant HIST1H1E is associated with altered methylation patterns . The antibody can be used in conjunction with methylation analyses to explore this connection.

Researchers should note that if mutations affect the epitope around Thr145, antibody recognition might be compromised, potentially necessitating antibodies targeting different regions of the protein.

What is known about HIST1H1E in cancer research?

HIST1H1E has several connections to cancer biology that can be investigated using the HIST1H1E (Ab-145) antibody:

• Mutations in Lymphomas: HIST1H1E and other H1 variants (H1B, H1C, H1D) exhibit diverse missense mutations in diffuse large B-cell lymphoma, Hodgkin lymphoma, and follicular lymphoma . These mutations can lead to "loss of nucleosome association and/or reduced capacity for chromatin compaction" .

• Expression Analysis: The antibody can be used in immunohistochemistry or Western blot analyses to compare HIST1H1E expression between normal and cancerous tissues.

• Chromatin Organization: Cancer cells frequently display altered chromatin organization. The antibody can help visualize differences in HIST1H1E distribution and chromatin structure between normal and cancer cells.

• Epigenetic Regulation: HIST1H1E plays a role in epigenetic regulation, which is often dysregulated in cancer. The antibody can be used to study how HIST1H1E-mediated epigenetic mechanisms are altered in cancer cells.

How can researchers validate HIST1H1E (Ab-145) antibody specificity?

To ensure experimental rigor, researchers should validate the specificity of the HIST1H1E (Ab-145) antibody through several approaches:

• Positive and Negative Controls:

  • Positive control: Cell lines known to express HIST1H1E (e.g., HeLa cells)

  • Negative control: CRISPR/Cas9-mediated HIST1H1E knockout cells

  • Peptide competition assay using the immunizing peptide

• Cross-Reactivity Testing:

  • Western blot analysis using recombinant H1 variants to assess potential cross-reactivity

  • Comparison with other validated HIST1H1E antibodies targeting different epitopes

• Application-Specific Validation:

  • For ChIP: Include IgG control and validate enrichment at known binding sites

  • For immunofluorescence: Compare with other nuclear markers and assess expected nuclear localization

How can HIST1H1E (Ab-145) antibody be used in studying immune cell differentiation?

Recent research has identified an important role for linker histones, particularly H1.2 and H1.4, in immune cell differentiation . The HIST1H1E (Ab-145) antibody can be valuable for studying these processes:

• Neutrophil Differentiation: H1.4 (HIST1H1E) has been shown to affect neutrophil lineage determination . The antibody can be used to track H1.4 expression and localization during neutrophil differentiation.

• Expression Dynamics: Research indicates that "PLB-985 expressed mRNA encoding H1.2 and H1.4 more abundantly than the other subtypes" and that "protein levels also decreased as the cells matured" . The antibody can be used to confirm these findings at the protein level.

• Lineage Specification: H1 subtypes affect lineage specification during granulopoiesis, with loss of H1.2 and H1.4 inducing an eosinophil-like transcriptional program . The antibody can help investigate how H1.4 levels correlate with cell fate decisions.

• GATA-2 Connection: The subtype-specific function of H1 in neutrophil and eosinophil differentiation depends partly on the transcription factor GATA-2 . Co-immunoprecipitation experiments using the antibody could help elucidate this relationship.

What are emerging applications for HIST1H1E (Ab-145) antibody?

Several emerging research areas could benefit from utilizing the HIST1H1E (Ab-145) antibody:

• Gene Therapy Development: As of 2024, research into HIST1H1E syndrome at Yale School of Medicine is aiming to develop gene-editing therapy that could be delivered to the brain, potentially correcting the function of histone H1.4 in neuronal cells .

• Single-Cell Epigenomics: The antibody could be adapted for CUT&Tag or other single-cell epigenomic profiling methods to study HIST1H1E distribution at the single-cell level.

• Liquid Biopsy Biomarkers: Given the links between histone variants and cancer, the antibody could potentially be used to detect circulating HIST1H1E or its fragments as potential biomarkers.

• Developmental Epigenetics: Since HIST1H1E mutations cause developmental disorders, the antibody could be valuable for studying the role of this histone in normal development and differentiation.

What methodological advances could enhance HIST1H1E studies?

Several methodological approaches could enhance research using the HIST1H1E (Ab-145) antibody:

• Proximity Labeling: Combining the antibody with proximity labeling techniques (BioID, APEX) could identify novel interaction partners of HIST1H1E.

• Live-Cell Imaging: Development of fluorescently tagged nanobodies based on the HIST1H1E (Ab-145) epitope could enable live-cell tracking of HIST1H1E dynamics.

• Mass Spectrometry Integration: Using the antibody for immunoprecipitation followed by mass spectrometry could identify post-translational modifications and protein interactions.

• CRISPR Screening: The antibody could be used to validate hits from CRISPR screens targeting regulators of HIST1H1E expression or function, similar to the genome-wide CRISPR/Cas9 screen that identified HIST1H1E's role in neutrophil differentiation .