HIST1H1E (Ab-17) Antibody

What is HIST1H1E and why is it significant in chromatin research?

HIST1H1E, also known as H1.4, is a linker histone protein that belongs to the H1 histone family. It plays a crucial role in chromatin compaction and organization by binding to nucleosomes and facilitating higher-order chromatin structure. HIST1H1E is particularly significant because it functions as a transcriptional repressor by limiting chromatin accessibility . Research has shown that HIST1H1E expression is 2-4 fold higher in germinal center B-cells compared to naïve B-cells, suggesting tissue-specific regulatory functions . The protein's role in genomic architecture makes it a critical target for epigenetic studies, particularly those examining chromatin remodeling and gene expression regulation.

What are the primary applications for the HIST1H1E (Ab-17) antibody?

The HIST1H1E (Ab-17) antibody has been validated for multiple research applications including:

• Western blotting (WB) for protein expression analysis

• Immunohistochemistry (IHC) for tissue localization studies

• Immunofluorescence (IF) for subcellular localization

• ELISA for quantitative protein detection

This polyclonal antibody recognizes human HIST1H1E (H1.4 linker histone), specifically targeting the region around the phosphorylation site at threonine 17 (Thr17) . The versatility of this antibody makes it valuable for researchers studying histone modifications, chromatin dynamics, and epigenetic regulation in both normal cellular processes and disease states.

What is the relationship between HIST1H1E mutations and disease?

Mutations in HIST1H1E have been identified as genetic drivers in several pathological conditions:

• B-cell lymphomas: Histone H1 mutations (including HIST1H1E) are recurrent in B-cell lymphomas and function as driver mutations. These mutations can lead to architectural remodeling of the genome with large-scale chromatin decompaction .

• Rahman syndrome: This rare congenital anomaly syndrome results from pathogenic variants in the HIST1H1E gene. Whole-exome sequencing has identified frameshift mutations in the C-terminal domain (CTD) of HIST1H1E as causative for this condition .

• Accelerated aging disorders: Frameshift mutations affecting the C-terminal tail of HIST1H1E have been associated with disruption of proper DNA compaction, reduced cellular proliferation, accelerated senescence, and premature aging phenotypes .

These disease associations highlight the critical importance of HIST1H1E in normal development and cellular homeostasis, making antibodies targeting this protein valuable tools for investigating disease mechanisms.

How should the HIST1H1E (Ab-17) antibody be optimized for Western blotting?

For optimal Western blotting results with the HIST1H1E (Ab-17) antibody, researchers should follow these methodological guidelines:

• Dilution range: Use at 1:500-1:2000 dilution for Western blotting applications .

• Sample preparation: Given that HIST1H1E is a nuclear protein tightly associated with chromatin, effective nuclear extraction protocols are essential. Use specialized nuclear extraction buffers containing DNase treatment to ensure complete release of histone proteins.

• Gel selection: Use 15-18% polyacrylamide gels to achieve proper separation of histone proteins, which have relatively low molecular weights.

• Blocking conditions: Use 5% BSA in TBST rather than milk, as milk contains casein which has phosphate groups that may interfere with phospho-specific antibody binding, particularly important since this antibody recognizes the phosphorylated Thr17 site .

• Detection system: Enhanced chemiluminescence (ECL) systems with high sensitivity are recommended due to the often low abundance of specific histone modifications.

• Positive controls: Include samples with known phosphorylation status at Thr17 of HIST1H1E to validate antibody specificity.

• Storage and handling: Store at -20°C or -80°C and avoid repeated freeze-thaw cycles to maintain antibody integrity .

What protocol should be followed for immunofluorescence studies with HIST1H1E (Ab-17) antibody?

For immunofluorescence applications using the HIST1H1E (Ab-17) antibody, follow this optimized protocol:

• Fixation: Fix cells using 4% paraformaldehyde for 15 minutes at room temperature to preserve nuclear structure.

• Permeabilization: Use 0.2% Triton X-100 for 10 minutes to allow antibody access to nuclear proteins.

• Blocking: Block with 3-5% BSA for 1 hour at room temperature.

• Primary antibody: Apply HIST1H1E (Ab-17) antibody at 1:200-1:1000 dilution in blocking buffer. Incubate overnight at 4°C.

• Washing: Perform 3-5 washes with PBS containing 0.1% Tween-20.

• Secondary antibody: Use fluorophore-conjugated anti-rabbit secondary antibodies (since this is a rabbit polyclonal antibody ).

• Counterstaining: DAPI staining for nuclear visualization is essential since HIST1H1E is a nuclear protein.

• Mounting and imaging: Use anti-fade mounting medium and confocal microscopy for high-resolution imaging of nuclear structures.

• Controls: Include negative controls (secondary antibody only) and positive controls (cells known to express HIST1H1E) to validate staining specificity.

When analyzing results, expect a nuclear localization pattern with potential variation in intensity depending on cell cycle phase, as histone phosphorylation states can change during mitosis.

What are the critical considerations for immunohistochemistry applications?

When performing immunohistochemistry (IHC) with the HIST1H1E (Ab-17) antibody, researchers should consider these methodological aspects:

• Tissue preparation: Proper fixation is crucial; use 10% neutral buffered formalin followed by paraffin embedding. Frozen sections may also be used but may show different antigen accessibility.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is recommended to expose epitopes masked during fixation.

• Dilution optimization: Start with 1:100-1:300 dilution as recommended , but optimize through titration experiments for each tissue type.

• Incubation conditions: Incubate primary antibody overnight at 4°C for optimal sensitivity.

• Detection system: Use polymer-based detection systems rather than avidin-biotin complexes to reduce background in nuclear staining.

• Chromogen selection: DAB (3,3'-diaminobenzidine) provides good contrast for nuclear proteins against hematoxylin counterstain.

• Counterstaining: Light hematoxylin counterstaining helps visualize nuclear morphology without obscuring antibody signal.

• Controls: Include tissues known to express HIST1H1E as positive controls. Tonsil tissue, which contains germinal center B-cells with high HIST1H1E expression , would be particularly appropriate.

• Interpreting results: Nuclear staining patterns should be evaluated for intensity and distribution, with attention to variation between different cell types and tissue regions.

How can HIST1H1E (Ab-17) antibody be used to study chromatin remodeling in lymphoma models?

The HIST1H1E (Ab-17) antibody can be a powerful tool for investigating chromatin remodeling in lymphoma models through these advanced methodological approaches:

• Chromatin immunoprecipitation (ChIP): Though not explicitly listed in the technical specifications, the antibody's ability to recognize HIST1H1E can be leveraged for ChIP experiments to map genome-wide binding patterns of HIST1H1E in normal versus lymphoma cells. This application would require optimization beyond standard protocols.

• Co-immunoprecipitation studies: Use the antibody to pull down HIST1H1E and identify interacting proteins that may be dysregulated in lymphoma, particularly focusing on chromatin-modifying enzymes.

• Comparative immunofluorescence: Perform quantitative immunofluorescence analysis comparing HIST1H1E distribution patterns between normal B-cells and lymphoma cells, looking for differences in nuclear localization and intensity that may correlate with chromatin decompaction observed in lymphomas with H1 mutations .

• Correlation with epigenetic marks: Combine with antibodies against H3K36me2 and H3K27me3 in multi-label immunofluorescence or sequential IHC to examine the relationship between HIST1H1E binding and these epigenetic marks, which are known to be altered in lymphomas with H1 mutations .

• Analysis of phosphorylation dynamics: Since the antibody specifically recognizes the Thr17 phosphorylation site , use it to track changes in HIST1H1E phosphorylation during lymphoma progression, particularly in relation to cell cycle phases and treatment responses.

• Mouse model validation: Use in studies of H1c/e-deficient mouse models that develop aggressive lymphomas with enhanced repopulating potential to confirm molecular mechanisms of lymphomagenesis.

What are the methodological approaches for investigating HIST1H1E in cellular senescence and aging?

To investigate the role of HIST1H1E in cellular senescence and aging, researchers can employ these methodological strategies with the HIST1H1E (Ab-17) antibody:

• Senescence marker correlation: Perform dual immunofluorescence with HIST1H1E (Ab-17) antibody and senescence markers (p16, p21, SA-β-gal) to examine correlations between HIST1H1E phosphorylation status and cellular senescence.

• Time-course experiments: Utilize Western blotting with the antibody to track changes in HIST1H1E expression and phosphorylation throughout replicative senescence in primary cell cultures.

• Genome-wide analysis: Combine ChIP using HIST1H1E (Ab-17) antibody with sequencing (ChIP-seq) to map changes in HIST1H1E binding patterns during senescence, particularly at genes associated with the senescence-associated secretory phenotype (SASP).

• Mutation models: Apply the antibody in cellular models expressing HIST1H1E frameshift mutations associated with accelerated aging to examine protein localization and chromatin binding compared to wild-type protein.

• Chromatin compaction assays: Use the antibody in combination with techniques measuring chromatin accessibility (such as ATAC-seq) to correlate HIST1H1E binding with changes in chromatin compaction during senescence.

• DNA methylation studies: Combine HIST1H1E immunoprecipitation with DNA methylation analysis to investigate the relationship between HIST1H1E binding and methylation profiles associated with aging, as frameshift mutations in HIST1H1E have been linked to specific methylation patterns .

• Intervention studies: Apply the antibody to assess changes in HIST1H1E phosphorylation and localization following interventions that extend cellular lifespan or reverse aspects of senescence.

How can researchers develop models to study Rahman syndrome using HIST1H1E (Ab-17) antibody?

Researchers developing models to study Rahman syndrome can utilize the HIST1H1E (Ab-17) antibody through these methodological approaches:

• Embryonic stem cell models: As described in the search results, mouse embryonic stem cell (mESC) models have been developed for Rahman syndrome using CRISPR/Cas9 genome engineering . The HIST1H1E (Ab-17) antibody can be used to:

  • Verify expression of mutant HIST1H1E protein

  • Compare localization patterns of wild-type versus mutant protein

  • Assess chromatin binding efficiency through chromatin fractionation followed by Western blotting

• Patient-derived cell analysis: For cells derived from Rahman syndrome patients (with frameshift mutations in the C-terminal domain of HIST1H1E ), the antibody can be used to:

  • Assess protein stability and nuclear localization

  • Evaluate phosphorylation status at Thr17

  • Compare expression levels between patient and control cells

• Differentiation studies: During neural differentiation of stem cell models (relevant to the neurodevelopmental aspects of Rahman syndrome), the antibody can track:

  • Changes in HIST1H1E expression and phosphorylation during differentiation

  • Correlation with neural marker expression

  • Differences in chromatin organization between wild-type and mutant cells

• Chromatin structure analysis: Combine the antibody with super-resolution microscopy techniques to visualize differences in higher-order chromatin structure between normal and Rahman syndrome model cells.

• Rescue experiments: In CRISPR-engineered models expressing tagged mutant HIST1H1E , use the antibody to distinguish between endogenous and mutant protein in rescue experiments.

What are common problems and solutions when using HIST1H1E (Ab-17) antibody in experimental applications?

Researchers may encounter several challenges when working with the HIST1H1E (Ab-17) antibody. Here are methodological solutions to common problems:

Problem 1: High background in Western blotting

• Solution: Increase blocking time to 2 hours at room temperature with 5% BSA, increase wash steps (5 x 5 minutes), and optimize antibody dilution (test range from 1:500 to 1:2000) .

• Additional approach: Use more stringent washing buffers (increase Tween-20 concentration to 0.1-0.2%) and consider alternative blocking agents such as fish gelatin.

Problem 2: Weak or no signal in IHC/IF

• Solution: Optimize antigen retrieval methods (try both citrate and EDTA-based buffers at different pH values and heating times).

• Additional approach: Extend primary antibody incubation time to overnight at 4°C and use signal amplification systems such as tyramide signal amplification.

Problem 3: Cross-reactivity with other histone H1 variants

• Solution: Include appropriate controls using tissues or cells with known expression patterns of different H1 variants.

• Additional approach: Consider validation with knockout or knockdown models of specific H1 variants to confirm specificity.

Problem 4: Inconsistent results across experiments

• Solution: Standardize tissue/cell processing protocols and antibody handling. Avoid repeated freeze-thaw cycles of the antibody .

• Additional approach: Prepare single-use aliquots of the antibody and store at recommended temperature (-20°C to -80°C) .

Problem 5: Difficulties detecting phosphorylated epitope

• Solution: Include phosphatase inhibitors in all sample preparation buffers and handle samples at 4°C to preserve phosphorylation status.

• Additional approach: Consider using phosphatase treatment as a negative control to validate phospho-specific detection.

How should researchers validate the specificity of HIST1H1E (Ab-17) antibody?

Thorough validation of antibody specificity is crucial for reliable research results. For the HIST1H1E (Ab-17) antibody, implement these methodological validation approaches:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (derived from Human Histone H1.4 around Thr17 ) to confirm that this blocks specific binding in Western blot or IHC applications.

• Genetic validation: Test the antibody in:

  • HIST1H1E knockout or knockdown models to confirm signal loss

  • Cells expressing tagged HIST1H1E to confirm co-localization

  • The mouse embryonic stem cell model developed for Rahman syndrome studies that expresses labeled mutant H1-4

• Phosphorylation-specific validation: Treat samples with lambda phosphatase to remove phosphate groups and confirm loss of signal, validating the phospho-specificity of the antibody.

• Cross-reactivity testing: Test on samples from different species to confirm the stated cross-reactivity with human, monkey, and mouse samples .

• Western blot validation: Confirm that the antibody detects a band of the expected molecular weight (approximately 21.9 kDa) for HIST1H1E.

• Immunoprecipitation followed by mass spectrometry: Perform IP with the antibody followed by mass spectrometry to identify all proteins being pulled down, confirming specificity for HIST1H1E.

• Comparison with other validated HIST1H1E antibodies: Compare staining patterns with those obtained using other antibodies targeting different epitopes of HIST1H1E.

• Correlation with mRNA expression: Compare antibody signal intensity with HIST1H1E mRNA levels across different cell types or tissues to confirm biological relevance of the signal.

How can HIST1H1E (Ab-17) antibody contribute to understanding epigenetic reprogramming in cancer?

The HIST1H1E (Ab-17) antibody can be instrumental in advancing our understanding of epigenetic reprogramming in cancer through these methodological approaches:

• Mapping chromatin accessibility changes: Use the antibody in combination with ATAC-seq or DNase-seq to correlate HIST1H1E binding with changes in chromatin accessibility in cancer cells, particularly in lymphomas where H1 mutations lead to chromatin decompaction .

• Investigating developmental gene reactivation: Research has shown that loss of H1 proteins can "unlock expression of stem cell genes that are normally silenced during early development" . The antibody can be used to track HIST1H1E binding at these developmental gene loci in normal versus cancer cells.

• Monitoring treatment responses: Apply the antibody to monitor changes in HIST1H1E phosphorylation and localization in response to epigenetic therapies (HDAC inhibitors, DNA methyltransferase inhibitors) to understand treatment mechanisms.

• Biomarker development: Evaluate HIST1H1E phosphorylation at Thr17 as a potential prognostic or predictive biomarker in cancer tissues using quantitative IHC or tissue microarrays.

• Three-dimensional genome organization: Combine with Hi-C or other 3D genome mapping techniques to understand how HIST1H1E contributes to maintaining genomic compartmentalization and how this is disrupted in cancer.

• Single-cell analysis: Adapt the antibody for single-cell proteomics or CyTOF analysis to understand heterogeneity in HIST1H1E expression and phosphorylation within tumor populations.

• Therapeutic targeting: Use the antibody to validate the effects of experimental compounds designed to modulate histone H1 function as potential cancer therapeutics.

What experimental approaches can reveal the relationship between HIST1H1E phosphorylation and chromatin dynamics?

To investigate the relationship between HIST1H1E phosphorylation and chromatin dynamics, researchers can employ these advanced methodological strategies using the HIST1H1E (Ab-17) antibody:

• Live-cell imaging: Combine with fluorescently tagged chromatin markers to correlate HIST1H1E phosphorylation status with chromatin compaction states in real-time.

• FRAP (Fluorescence Recovery After Photobleaching): Use in conjunction with GFP-tagged HIST1H1E to measure how phosphorylation at Thr17 affects the mobility and chromatin binding dynamics of HIST1H1E.

• Proximity ligation assays (PLA): Combine the HIST1H1E (Ab-17) antibody with antibodies against other chromatin-associated proteins to detect and quantify protein-protein interactions dependent on phosphorylation status.

• Super-resolution microscopy: Apply techniques like STORM or PALM to visualize nanoscale changes in chromatin organization associated with HIST1H1E phosphorylation at Thr17.

• Targeted phosphatase/kinase experiments: Identify and modulate the enzymes responsible for Thr17 phosphorylation/dephosphorylation and use the antibody to track resulting changes in chromatin structure.

• Cell-cycle synchronization studies: Use the antibody to track changes in HIST1H1E phosphorylation throughout different cell cycle phases and correlate with chromatin condensation states.

• In vitro reconstitution: Perform in vitro chromatin assembly assays with recombinant nucleosomes and either phosphorylated or non-phosphorylated HIST1H1E, using the antibody to confirm phosphorylation status.

• Cryo-electron microscopy: Use immunogold labeling with the antibody to visualize HIST1H1E phosphorylation in the context of higher-order chromatin structures.