HIST1H2BC (Ab-5) Antibody

What is HIST1H2BC and what cellular functions does it regulate?

HIST1H2BC is a core component of the nucleosome that plays a central role in DNA packaging into chromatin. As part of the histone H2B family, it functions as a structural protein that limits DNA accessibility to cellular machinery requiring DNA as a template. This regulation significantly impacts transcription, DNA repair, DNA replication, and chromosomal stability . The protein belongs to a family of highly similar histone variants including HIST1H2BC/E/F/G/I, which share identical amino acid sequences despite having distinct nucleotide sequences . These variants collectively contribute to the epigenetic regulation of gene expression through their incorporation into chromatin.

How does HIST1H2BC (Ab-5) antibody differ from other H2B antibodies?

The HIST1H2BC (Ab-5) antibody specifically targets the region around the lysine 5 residue of Histone H2B type 1-C/E/F/G/I, making it distinct from other antibodies such as HIST1H2BC (Ab-108) which targets the region around lysine 108 . This epitope specificity determines which post-translational modifications the antibody can detect, as lysine 5 may undergo different modifications compared to other residues. The antibody is raised in rabbits as a polyclonal preparation and purified using antigen affinity methods . Unlike pan-H2B antibodies that recognize all H2B variants, the HIST1H2BC (Ab-5) targets specific variants, allowing researchers to distinguish between closely related histone proteins in experimental analyses.

What are the recommended experimental conditions for using HIST1H2BC (Ab-5) antibody in immunofluorescence studies?

For immunofluorescence (IF) applications, the HIST1H2BC (Ab-5) antibody should be used at a dilution range of 1:1-1:10 . The following protocol is recommended:

• Fix cells/tissues with 4% paraformaldehyde for 10 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 in PBS for 5 minutes

• Block with 5% normal serum in PBS containing 0.1% Tween-20 for 1 hour

• Incubate with HIST1H2BC (Ab-5) antibody at the recommended dilution overnight at 4°C

• Wash 3x with PBS-T (PBS with 0.1% Tween-20)

• Incubate with fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI

• Mount and image

When interpreting results, researchers should compare signal patterns with control samples and consider co-localization with other nuclear markers to confirm specificity of nuclear staining patterns.

How can I optimize HIST1H2BC (Ab-5) antibody for immunohistochemistry in tissue sections?

For immunohistochemistry (IHC) applications, the HIST1H2BC (Ab-5) antibody requires optimization with the following methodological considerations:

• Dilution range: Start with 1:10-1:100 as recommended , then titrate for optimal signal-to-noise ratio

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is typically most effective for histone proteins

• Blocking: Use 5-10% normal serum from the species of the secondary antibody

• Incubation time: Overnight at 4°C generally yields best results

• Detection system: Polymer-based detection systems may provide better sensitivity than ABC methods

• Counterstaining: Light hematoxylin counterstaining to visualize nuclei without obscuring antibody signal

Tissue-specific optimization may be necessary, as histone accessibility can vary between different fixation protocols and tissue types. Positive controls should include tissues known to express H2B histones abundantly, such as testis or thymus tissue, which show high expression of related H2B variants .

What are the most effective protein extraction methods for detecting HIST1H2BC in Western blot applications?

While HIST1H2BC (Ab-5) antibody is not specifically recommended for Western blot in the provided information, related H2B antibodies have been used successfully in this application . For optimal histone extraction for Western blotting:

• Acid Extraction Method:

  • Harvest cells and wash with ice-cold PBS

  • Resuspend cell pellet in 0.2M H₂SO₄

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 16,000g for 10 minutes at 4°C

  • Transfer supernatant to a new tube

  • Precipitate histones by adding TCA to final concentration of 33%

  • Incubate on ice for 30 minutes

  • Centrifuge at 16,000g for 10 minutes at 4°C

  • Wash pellet twice with ice-cold acetone

  • Air-dry pellet and dissolve in appropriate buffer

• Nuclear Extraction with High Salt:

  • Lyse cells in hypotonic buffer to isolate nuclei

  • Extract histones from nuclei using high-salt buffer (400-500mM NaCl)

  • Remove debris by centrifugation

When analyzing Western blot results, note that variant-specific antibodies like HIST1H2BC (Ab-5) may not show strong signals in total protein extracts due to the relatively small contribution of specific variants to the total histone pool .

How should I troubleshoot weak or absent signals when using HIST1H2BC (Ab-5) antibody in ELISA assays?

For ELISA applications, where the recommended dilution is not specified in the provided material but can be extrapolated from similar antibodies to be around 1:2000-1:10000 , consider the following troubleshooting steps for weak signals:

• Antibody concentration: Increase antibody concentration by using a lower dilution

• Antigen concentration: Ensure sufficient antigen coating on the plate

• Blocking optimization: Test different blocking agents (BSA, milk, serum)

• Incubation conditions: Extend incubation time or optimize temperature

• Detection system sensitivity: Switch to a more sensitive detection method

• Buffer composition: Ensure optimal pH and salt concentration for antibody-antigen interaction

• Sample preparation: Check if target protein is denatured/modified in a way that affects epitope recognition

If multiple approaches fail to improve signal, consider that HIST1H2BC levels may be inherently low in your samples, as variant expression is tissue-specific and can vary significantly between different cell types .

How can HIST1H2BC (Ab-5) antibody be used to investigate histone degradation pathways in nutrient-deprived conditions?

Recent research has revealed that histone H2B proteins undergo global degradation in response to environmental stressors such as nutrient deprivation . To investigate this process using HIST1H2BC (Ab-5) antibody:

• Experimental design:

  • Culture cells under normal and nutrient-deprived conditions

  • Harvest cells at multiple time points (0, 2, 4, 8, 12, 24 hours)

  • Perform immunofluorescence using HIST1H2BC (Ab-5) antibody

  • Quantify nuclear fluorescence intensity

• Co-immunoprecipitation approach:

  • Use HIST1H2BC (Ab-5) antibody to pull down H2B and associated proteins

  • Probe for ubiquitination using anti-K48-linked polyubiquitin antibodies

  • Identify interacting E2/E3 ligases through mass spectrometry

• Proteasome inhibitor studies:

  • Treat cells with proteasome inhibitors (e.g., MG-132)

  • Compare H2B levels between treated and untreated samples using immunofluorescence

  • Investigate accumulation of ubiquitinated forms

Research suggests that K31 mediates H2B polyubiquitination and degradation . Examining how starvation affects HIST1H2BC levels compared to replication-independent H2B variants could provide insights into differential regulation of histone variants during cellular stress.

What is the significance of HIST1H2BC in epigenetic regulation of cancer, and how can the antibody be used to study this relationship?

HIST1H2BC and related variants have been implicated in cancer progression, particularly in endocrine-resistant breast cancer . To investigate their role:

• Chromatin immunoprecipitation (ChIP) analysis:

  • Use HIST1H2BC (Ab-5) antibody to immunoprecipitate chromatin

  • Sequence associated DNA to identify genomic regions enriched for this variant

  • Compare binding patterns between normal and cancer cells

• Expression correlation studies:

  • Analyze HIST1H2BC expression in cancer cell lines using immunofluorescence

  • Correlate expression levels with cancer phenotypes and treatment responses

  • Compare with other histone variants to identify cancer-specific patterns

• DNA methylation analysis:

  • Investigate relationship between HIST1H2BC incorporation and DNA methylation

  • Studies have shown hypomethylation of related H2B variants in resistant cancer cells

  • Combine with bisulfite sequencing to correlate histone variant presence with methylation status

The HIST1H2BC (Ab-5) antibody can be particularly valuable for examining whether specific post-translational modifications near lysine 5 are associated with cancer progression or treatment resistance.

How can I design experiments to investigate the relationship between HIST1H2BC post-translational modifications and insulin/IGF signaling?

Research has identified connections between H2B degradation and insulin/IGF signaling pathways . To explore this relationship using HIST1H2BC (Ab-5) antibody:

• Signaling pathway intervention:

  • Treat cells with insulin pathway activators or inhibitors

  • Monitor changes in HIST1H2BC levels and modifications

  • Use immunofluorescence to quantify nuclear levels

• Analysis of lysine 5 modifications:

  • The HIST1H2BC (Ab-5) antibody targets the region around lysine 5

  • Design co-localization studies with antibodies against specific modifications (acetylation, methylation)

  • Compare modification patterns between normal and insulin-resistant models

• Genetic manipulation approach:

  • Generate lysine-to-alanine mutants (K5A) to prevent modification

  • Compare cellular response to insulin stimulation

  • Assess downstream signaling pathway activation

• Proteomic identification of interacting partners:

  • Immunoprecipitate HIST1H2BC using the antibody

  • Perform mass spectrometry to identify binding partners

  • Look specifically for insulin signaling pathway components

This research could provide insights into how histone modifications at specific residues contribute to metabolic regulation and insulin response at the epigenetic level.

How do I design experiments to distinguish between highly similar H2B variants (HIST1H2BC/E/F/G/I) that share identical amino acid sequences?

Distinguishing between H2B variants with identical amino acid sequences presents a significant challenge. Consider these methodological approaches:

• mRNA expression analysis:

  • Design variant-specific primers targeting the nucleotide differences

  • Perform qRT-PCR to measure relative expression levels

  • Compare expression patterns across tissues or experimental conditions

• ChIP-seq with variant-specific antibodies:

  • Use antibodies targeting unique post-translational modifications

  • Compare genomic distribution patterns

  • Analyze associated regulatory elements

• Promoter activity studies:

  • Clone the promoter regions of each variant

  • Use reporter assays to measure differential regulation

  • Identify transcription factors that selectively regulate each variant

The table below shows relative expression of H2B variants across tissues, which can guide experimental design by selecting appropriate positive control tissues:

This expression pattern information is extrapolated from the research findings on tissue-specific expression .

What are the methodological considerations for studying HIST1H2BC in the context of chromatin remodeling during cellular differentiation?

Chromatin remodeling involving HIST1H2BC plays crucial roles during cellular differentiation. To investigate this process:

• Time-course analysis:

  • Collect samples at different stages of differentiation

  • Perform immunofluorescence with HIST1H2BC (Ab-5) antibody

  • Quantify changes in nuclear distribution patterns

• Co-localization with chromatin remodelers:

  • Combine HIST1H2BC staining with antibodies against chromatin remodeling complexes

  • Analyze spatial relationships during differentiation

  • Quantify co-localization coefficients at different timepoints

• Accessibility analysis:

  • Combine HIST1H2BC ChIP with ATAC-seq

  • Correlate HIST1H2BC occupancy with chromatin accessibility

  • Identify differentially accessible regions during differentiation

• Interaction with lineage-specific transcription factors:

  • Perform sequential ChIP (re-ChIP) with HIST1H2BC (Ab-5) and lineage-specific factors

  • Identify genomic regions co-occupied by both proteins

  • Correlate with gene expression changes during differentiation

Understanding how HIST1H2BC contributes to chromatin architecture changes during differentiation could provide insights into developmental processes and potential therapeutic targets for differentiation disorders.

How should I interpret conflicting results between HIST1H2BC protein and mRNA expression levels in my samples?

Discrepancies between mRNA and protein levels of HIST1H2BC are common and can arise from several factors:

• Post-transcriptional regulation:

  • Histone mRNAs are subject to specific degradation mechanisms

  • Analyze stability of HIST1H2BC mRNA using actinomycin D chase experiments

  • Compare half-lives between experimental conditions

• Post-translational regulation:

  • H2B histones undergo ubiquitin-mediated degradation

  • Test if proteasome inhibitors normalize the protein/mRNA ratio

  • Investigate specific E2/E3 ligases (e.g., UBE2K/UBC-20 or HECTD1/HECT-1)

• Dilution effect in total H2B pool:

  • Variant-specific changes may not be detectable in total H2B measurements

  • Compare with other variant-specific antibodies

  • Use targeted mass spectrometry for absolute quantification

• Technical considerations:

  • Confirm antibody specificity with appropriate controls

  • Validate RT-PCR primers for specificity against highly similar variants

  • Consider histone extraction efficiency in different sample types

Research has shown that specific H2B variants can be significantly regulated without detectable changes in total H2B levels , highlighting the importance of variant-specific analysis.

What statistical approaches are most appropriate for analyzing HIST1H2BC immunofluorescence intensity data across experimental conditions?

When quantifying immunofluorescence data from HIST1H2BC (Ab-5) antibody staining:

• Image acquisition standardization:

  • Use identical exposure settings across all samples

  • Include calibration standards in each imaging session

  • Perform background subtraction consistently

• Quantification approaches:

  • Measure nuclear mean fluorescence intensity (MFI)

  • Consider integrated density (area × mean intensity) for total nuclear content

  • Analyze distribution patterns using intensity histograms

• Statistical analysis selection:

  • For normally distributed data: ANOVA with post-hoc tests for multiple comparisons

  • For non-parametric data: Kruskal-Wallis with Dunn's post-test

  • For time-course experiments: repeated measures ANOVA or mixed-effects models

• Visualization methods:

  • Box plots showing median and distribution

  • Violin plots for revealing distribution patterns

  • Scatter plots with individual data points for transparency

• Sample size considerations:

  • Minimum of 50-100 cells per condition for adequate statistical power

  • Account for biological replicates (different experiments)

  • Use power analysis to determine appropriate sample size

Careful statistical design is essential when analyzing subtle changes in histone variant distribution, particularly when examining heterogeneous cell populations.

How might HIST1H2BC (Ab-5) antibody contribute to research on histone variant exchange during cellular stress responses?

The HIST1H2BC (Ab-5) antibody offers unique opportunities to investigate histone variant dynamics during stress:

• Live-cell imaging applications:

  • Combine with GFP-tagged variants for FRAP (Fluorescence Recovery After Photobleaching)

  • Monitor incorporation rates under different stress conditions

  • Correlate with chromatin accessibility changes

• Nutrient deprivation studies:

  • Research has shown that replication-dependent H2B variants are replaced during starvation

  • Compare HIST1H2BC dynamics with replication-independent variants

  • Investigate tissue-specific responses to metabolic stress

• Therapeutic implications:

  • Examine how histone deacetylase inhibitors affect HIST1H2BC levels

  • Investigate synergistic effects with nutrient deprivation

  • Explore potential for targeting stress-specific histone exchange

• Single-cell analysis:

  • Develop immunofluorescence protocols compatible with single-cell sequencing

  • Correlate HIST1H2BC levels with transcriptional heterogeneity

  • Identify cell subpopulations with distinct stress responses

This research direction could reveal how chromatin organization adapts to environmental changes through selective incorporation of specific histone variants, with potential implications for stress response and adaptation mechanisms.

What technological advances might improve the specificity and sensitivity of HIST1H2BC detection in complex biological samples?

Emerging technologies could enhance HIST1H2BC detection:

• Proximity ligation assays (PLA):

  • Combine HIST1H2BC (Ab-5) with antibodies against specific modifications

  • Detect specific modified forms with higher sensitivity

  • Visualize spatial relationships between HIST1H2BC and interacting proteins

• Mass cytometry (CyTOF):

  • Develop metal-conjugated HIST1H2BC antibodies

  • Simultaneously measure multiple histone variants and modifications

  • Correlate with cellular phenotypes at single-cell resolution

• Super-resolution microscopy:

  • Visualize sub-nuclear distribution patterns of HIST1H2BC

  • Examine co-localization with chromatin features at nanometer resolution

  • Track dynamic changes during cellular processes

• Targeted proteomics:

  • Develop specific mass spectrometry assays for HIST1H2BC detection

  • Quantify variant-specific post-translational modifications

  • Measure absolute abundance in complex samples