HIST1H2BC (Ab-120) Antibody

What is the HIST1H2BC (Ab-120) Antibody and what specific epitope does it recognize?

HIST1H2BC (Ab-120) Antibody is a rabbit polyclonal antibody that specifically recognizes the peptide sequence surrounding lysine 120 (K120) in human Histone H2B type 1-C/E/F/G/I. This antibody targets one of the most functionally significant residues in the H2B family, as lysine 120 is a critical site for post-translational modifications, particularly ubiquitination and acetylation, which regulate chromatin structure and gene expression . The antibody demonstrates high specificity and sensitivity for human and rat samples, making it valuable for studying histone modifications in diverse experimental systems.

What validated applications are available for the HIST1H2BC (Ab-120) Antibody?

The HIST1H2BC (Ab-120) Antibody has been validated for multiple experimental applications, including:

• Enzyme-Linked Immunosorbent Assay (ELISA) - Recommended dilution range: 1:2000-1:10000

• Immunohistochemistry (IHC) - Recommended dilution range: 1:10-1:100

• Western Blot (WB) - For detection of histone H2B variants and their modifications

The antibody has been particularly validated for paraffin-embedded tissue sections, as demonstrated by its successful application in human breast cancer tissue with a Leica BondTM system after antigen retrieval under high pressure in citrate buffer (pH 6.0) . For optimal results in each application, antibody concentration should be empirically determined, as lot-specific variations may occur.

How should HIST1H2BC (Ab-120) Antibody be stored and handled to maintain its activity?

For optimal preservation of activity, the HIST1H2BC (Ab-120) Antibody should be stored in its liquid form in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . The antibody should be maintained at -20°C for long-term storage, and aliquoting is recommended to avoid repeated freeze-thaw cycles that can compromise antibody integrity. When handling for experiments, minimize time at room temperature and return to -20°C promptly after use. The antibody remains stable for at least 12 months when stored under these conditions. Centrifuge briefly before opening the vial to ensure collection of all material.

What is the recommended protocol for using HIST1H2BC (Ab-120) Antibody in immunohistochemistry?

For immunohistochemistry applications with paraffin-embedded tissues:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Deparaffinization and Rehydration:

  • Heat slides at 60°C for 1 hour

  • Deparaffinize in xylene (2 changes, 5 minutes each)

  • Rehydrate through graded alcohols to water

• Antigen Retrieval (Critical Step):

  • Perform high-pressure antigen retrieval in citrate buffer (pH 6.0)

  • This step is essential for exposing the epitope at lysine 120

• Blocking and Primary Antibody:

  • Block with 10% normal goat serum for 30 minutes

  • Apply HIST1H2BC (Ab-120) antibody at 1:10 dilution

  • Incubate overnight at 4°C or 1-2 hours at room temperature

• Detection and Visualization:

  • Use appropriate detection system (e.g., HRP-conjugated secondary antibody)

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Mount and evaluate under microscope

Validation experiments have shown successful staining in human breast cancer tissues using this protocol, with specific nuclear localization patterns consistent with histone proteins.

How can I detect H2BK120 ubiquitination using the HIST1H2BC (Ab-120) Antibody?

Detection of H2BK120 ubiquitination requires a strategic approach combining the HIST1H2BC (Ab-120) Antibody with other techniques:

• Cell/Tissue Preparation:

  • Harvest cells or prepare tissue lysates in the presence of deubiquitinase inhibitors (e.g., N-ethylmaleimide) to preserve ubiquitinated histones

  • Consider including proteasome inhibitors (e.g., MG132) in cell culture before harvesting

• Histone Extraction:

  • Extract histones using acid extraction method (0.2N HCl) to enrich histone proteins

  • Alternatively, use commercial histone extraction kits

• Western Blot Analysis:

  • Run extracted histones on 15% SDS-PAGE

  • Transfer to PVDF membrane (0.2 μm pore size recommended)

  • Block with 5% BSA or milk

• Antibody Detection Strategy:

  • Option 1: Use HIST1H2BC (Ab-120) Antibody to detect total H2B, then strip and reprobe with anti-ubiquitin antibody

  • Option 2: Perform immunoprecipitation with HIST1H2BC (Ab-120) Antibody, then blot for ubiquitin

  • Option 3: Use specific anti-H2BK120ub antibodies in parallel with HIST1H2BC (Ab-120) Antibody

• Validation Controls:

  • Include samples treated with deubiquitinating enzymes as negative controls

  • Include engineered H2BK120R mutant cells (where ubiquitination is prevented) as controls

This approach allows for specific detection of the ubiquitinated form of H2B at lysine 120, which is associated with active transcription and stimulation of H3K4 and H3K79 methylation.

What are the best methods for quantifying histone H2B variants using HIST1H2BC (Ab-120) Antibody?

Quantification of H2B variants requires specialized approaches due to their high sequence similarity:

• Mass Spectrometry-Based Quantification:

  • Perform top-down ultrahigh resolution Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry

  • This approach can distinguish between highly similar H2B variants based on small mass differences

  • Use HIST1H2BC (Ab-120) Antibody for immunoprecipitation prior to MS analysis to enrich for H2B variants

• ChIP-seq Approach:

  • Perform chromatin immunoprecipitation using HIST1H2BC (Ab-120) Antibody

  • Sequence precipitated DNA to identify genomic regions associated with specific H2B variants

  • Compare with other histone modification ChIP-seq data (H2BK120ub, H3K4me3, etc.)

• Western Blot-Based Semi-Quantification:

  • Use high-resolution gels (15-20% SDS-PAGE) to separate similar H2B variants

  • Transfer to 0.2 μm PVDF membrane

  • Probe with HIST1H2BC (Ab-120) Antibody

  • Use densitometry software for relative quantification

  • Include recombinant H2B variants as standards for calibration

For accurate variant identification, mass spectrometry approaches are preferred as they can resolve the small differences between H2B variants that differ by only a few amino acids.

How can HIST1H2BC (Ab-120) Antibody be used to study the relationship between H2BK120 ubiquitination and H3K79 methylation?

H2BK120 ubiquitination is known to stimulate H3K79 methylation by Dot1L, creating an important histone crosstalk mechanism. To investigate this relationship:

• Sequential ChIP (ChIP-reChIP):

  • First ChIP: Use HIST1H2BC (Ab-120) Antibody to pull down H2B-containing chromatin

  • Elute and perform second ChIP with anti-H2BK120ub antibody

  • Perform a third ChIP with anti-H3K79me2/3 antibodies

  • Analyze co-occupancy at specific genomic regions by qPCR or sequencing

• In Vitro Reconstitution Assay:

  • Prepare nucleosomes with either unmodified H2B or H2BK120ub (using dichloroacetone crosslinking method)

  • Incubate with recombinant Dot1L and S-adenosylmethionine (SAM)

  • Use HIST1H2BC (Ab-120) Antibody to immunoprecipitate the reaction products

  • Analyze H3K79 methylation levels by western blot with anti-H3K79me2/3 antibodies

• Structural Analysis:

  • Use HIST1H2BC (Ab-120) Antibody to validate the positioning of H2B in cryo-EM studies

  • Compare Dot1L binding to H2BK120ub nucleosomes versus unmodified nucleosomes

  • Investigate the structural basis of how H2BK120ub stimulates Dot1L activity

These approaches can reveal mechanistic insights into how H2BK120 ubiquitination facilitates H3K79 methylation, which is critical for gene activation and genomic stability.

How does H2BK120 acetylation influence BRD4 binding, and how can HIST1H2BC (Ab-120) Antibody be used to study this interaction?

Recent research has shown that acetylation of H2BK120 (H2BK120ac) regulates BRD4 binding specifically at intergenic enhancers (IGEs) but not at gene body enhancers (GBEs) or promoters. To study this:

• ChIP-seq Comparative Analysis:

  • Perform ChIP-seq with HIST1H2BC (Ab-120) Antibody to establish H2B distribution

  • Perform parallel ChIP-seq with anti-H2BK120ac and anti-BRD4 antibodies

  • Use cells expressing H2BK120R mutant (which cannot be acetylated) as controls

  • Compare binding patterns to identify regions where H2BK120ac correlates with BRD4 recruitment

• Machine Learning Approach:

  • Generate datasets of histone modification ChIP-seq signals

  • Use random forest regressor machine learning to identify histone acetylations that predict BRD4 binding

  • Validate findings using HIST1H2BC (Ab-120) Antibody in cells with H2BK120R mutation

• Sequential ChIP with Protein Interaction Analysis:

  • Use HIST1H2BC (Ab-120) Antibody for initial ChIP

  • Perform secondary ChIP with anti-H2BK120ac

  • Analyze BRD4 recruitment using proximity ligation assay (PLA)

  • Compare results across different genomic contexts (IGEs vs. GBEs vs. promoters)

These methods can elucidate the context-specific role of H2BK120 acetylation in regulating BRD4 binding and the subsequent impact on transcriptional regulation.

What is the role of H2BK120 ubiquitination in regulating nucleosome acidic patch interactions, and how can this be studied?

H2BK120 ubiquitination functions as a gatekeeper for the nucleosome acidic patch, a critical binding surface for many chromatin-interacting proteins. To investigate this regulatory mechanism:

• Competitive Binding Assays:

  • Prepare nucleosomes with and without H2BK120ub modification

  • Use HIST1H2BC (Ab-120) Antibody to verify nucleosome quality

  • Perform binding assays with known acidic patch-binding proteins (e.g., RCC1, LANA)

  • Compare binding affinities to determine how H2BK120ub affects protein interactions

• Cryo-EM Structural Analysis:

  • Generate H2BK120ub nucleosomes using chemical crosslinking approaches

  • Validate with HIST1H2BC (Ab-120) Antibody

  • Perform cryo-EM to visualize ubiquitin positioning relative to the acidic patch

  • Compare with H2AK119ub nucleosomes to highlight distinct mechanisms

• Functional Genomics Approach:

  • Express H2BK120R mutant to prevent ubiquitination

  • Perform ChIP-seq for acidic patch-binding proteins

  • Correlate with transcriptional changes and chromatin accessibility

  • Use HIST1H2BC (Ab-120) Antibody to validate H2B levels across conditions

These approaches can reveal how H2BK120ub selectively regulates protein interactions at the nucleosome acidic patch, providing insights into its role in gene regulation and chromatin organization.

What are common technical challenges when using HIST1H2BC (Ab-120) Antibody, and how can they be addressed?

Several technical challenges may arise when working with HIST1H2BC (Ab-120) Antibody:

• Cross-reactivity with other H2B variants:

  • Challenge: Due to high sequence similarity between H2B variants, cross-reactivity may occur

  • Solution: Perform antibody validation using recombinant H2B variants or H2B variant-depleted samples

  • Methodology: Use knockout/knockdown verification or peptide competition assays to confirm specificity

• Accessing the epitope in fixed tissues:

  • Challenge: Formalin fixation may mask the K120 epitope

  • Solution: Optimize antigen retrieval methods

  • Methodology: Test different buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0) and retrieval methods (heat-induced vs. enzymatic)

• Detecting low-abundance modifications:

  • Challenge: H2BK120 modifications may be present at low levels

  • Solution: Enrich for modified histones prior to antibody application

  • Methodology: Use PTM-specific enrichment techniques (e.g., ubiquitin affinity resins) followed by detection with HIST1H2BC (Ab-120) Antibody

• High background in IHC applications:

  • Challenge: Non-specific staining in certain tissues

  • Solution: Optimize blocking conditions and antibody dilution

  • Methodology: Test different blocking reagents (BSA, normal serum, commercial blockers) and titrate antibody concentration

Careful optimization of these parameters will ensure specific and sensitive detection of H2B and its K120-modified forms.

How can I distinguish between different histone H2B variants when using HIST1H2BC (Ab-120) Antibody?

Distinguishing between highly similar H2B variants requires specialized approaches:

• Two-dimensional gel electrophoresis:

  • First dimension: Separate by isoelectric point

  • Second dimension: Separate by molecular weight

  • Western blot with HIST1H2BC (Ab-120) Antibody

  • Compare spot patterns with known variant standards

• Mass spectrometry validation:

  • Immunoprecipitate with HIST1H2BC (Ab-120) Antibody

  • Analyze by top-down mass spectrometry

  • Identify variants based on unique peptide signatures

  • Compare with theoretical masses of known H2B variants

• Variant-specific RNA expression correlation:

  • Perform RT-qPCR to quantify expression levels of specific H2B variant transcripts

  • In parallel, perform protein analysis with HIST1H2BC (Ab-120) Antibody

  • Correlate protein levels with transcript abundance

  • Use cells with manipulated H2B variant expression as controls

These complementary approaches can help resolve the complex landscape of H2B variants, which is particularly important in cancer research where specific variants show altered expression patterns.

How can HIST1H2BC (Ab-120) Antibody be used in multiplexed imaging approaches to study histone modifications?

Multiplexed imaging of histone modifications enables visualization of multiple epigenetic marks simultaneously:

• Sequential Immunofluorescence:

  • First round: Stain with HIST1H2BC (Ab-120) Antibody and fluorescently labeled secondary antibody

  • Image and record positions

  • Strip antibodies using glycine buffer (pH 2.5) or commercial antibody stripping solutions

  • Stain with antibodies against other histone modifications (H3K4me3, H3K79me2, H2BK120ub)

  • Overlay images to analyze co-localization patterns

• Multiplexed Immunohistochemistry:

  • Use tyramide signal amplification (TSA) system

  • Label HIST1H2BC (Ab-120) Antibody with one fluorophore

  • Label other histone modification antibodies with different fluorophores

  • Sequential staining with microwave treatment between rounds

  • Analyze cellular and subcellular co-localization of histone marks

• Mass Cytometry (CyTOF):

  • Label HIST1H2BC (Ab-120) Antibody with a specific metal isotope

  • Label other antibodies with different metal isotopes

  • Analyze single cells for multiple histone modifications simultaneously

  • Correlate H2B levels with specific modifications and cellular phenotypes

These multiplexed approaches provide spatial information about histone modifications within the nucleus, enabling studies of their relationships in different chromatin domains and cellular states.

How can HIST1H2BC (Ab-120) Antibody be used to study histone H2B modifications in cancer?

The HIST1H2BC (Ab-120) Antibody offers valuable tools for investigating histone H2B's role in cancer:

• Cancer-specific H2B variant expression analysis:

  • Compare H2B variant profiles between normal and cancer tissues using HIST1H2BC (Ab-120) Antibody

  • Correlate with top-down mass spectrometry to identify cancer-specific variants

  • Track changes in H2B variants during cancer progression and in response to treatment

• H2BK120ub status in tumor samples:

  • Perform IHC on tissue microarrays from different tumor types

  • Use HIST1H2BC (Ab-120) Antibody alongside H2BK120ub-specific antibodies

  • Correlate H2BK120ub levels with:

    • Clinical outcomes

    • Tumor grade and stage

    • Expression of RNF20/40 (E3 ligases for H2BK120ub)

    • DNA damage response markers

• Epigenetic profiling of oncohistones:

  • Identify H2B variants acting as oncohistones in specific cancers

  • Use HIST1H2BC (Ab-120) Antibody to detect expression patterns

  • Perform ChIP-seq to map genomic distribution

  • Correlate with gene expression changes and cancer phenotypes

These approaches can reveal cancer-specific alterations in H2B variants and their modifications, potentially identifying new diagnostic markers or therapeutic targets.

What is the role of H2BK120 modifications in stem cell differentiation, and how can it be studied?

H2BK120 ubiquitination plays a critical role in embryonic stem cell differentiation:

• Temporal analysis during differentiation:

  • Track H2BK120ub levels during stem cell differentiation using western blot

  • Use HIST1H2BC (Ab-120) Antibody to normalize for total H2B

  • Correlate changes with differentiation markers and cell fate decisions

  • Compare with other histone modifications (H3K4me3, H3K79me2)

• Genomic redistribution analysis:

  • Perform ChIP-seq with HIST1H2BC (Ab-120) Antibody and H2BK120ub-specific antibodies

  • Compare genomic distribution in pluripotent and differentiated states

  • Analyze association with:

    • Pluripotency genes

    • Lineage-specific genes

    • Bivalent domains (H3K4me3/H3K27me3)

• Functional manipulation studies:

  • Generate stem cells expressing H2BK120R mutant (preventing ubiquitination)

  • Assess differentiation potential and lineage specification

  • Use HIST1H2BC (Ab-120) Antibody to confirm equal H2B expression

  • Rescue experiments with wild-type H2B reintroduction

These approaches can illuminate how H2BK120 modifications regulate the balance between self-renewal and differentiation in stem cells, with implications for regenerative medicine and developmental biology.

How might HIST1H2BC (Ab-120) Antibody contribute to understanding the structural and functional differences between H2B variants?

Future research utilizing HIST1H2BC (Ab-120) Antibody could explore:

• Variant-specific chromatin dynamics:

  • Combine HIST1H2BC (Ab-120) Antibody with super-resolution microscopy

  • Track real-time dynamics of different H2B variants in living cells

  • Correlate variant distribution with chromatin accessibility and transcriptional activity

  • Develop variant-specific antibodies based on HIST1H2BC (Ab-120) epitope mapping

• Cross-talk between H2B variants and other histone modifications:

  • Use HIST1H2BC (Ab-120) Antibody for sequential ChIP with antibodies against other modifications

  • Identify variant-specific modification patterns

  • Map these patterns genome-wide in different cell types and disease states

  • Develop computational models to predict functional outcomes

• H2B variant-specific protein interactions:

  • Perform proximity-dependent biotinylation (BioID) coupled with HIST1H2BC (Ab-120) immunoprecipitation

  • Identify proteins that preferentially interact with specific H2B variants

  • Validate interactions using in vitro reconstituted nucleosomes

  • Determine functional consequences of variant-specific interactions

These approaches could reveal previously unrecognized functions of H2B variants in chromatin organization and gene regulation.

What emerging techniques might enhance the utility of HIST1H2BC (Ab-120) Antibody in epigenetic research?

Emerging technologies could expand the applications of HIST1H2BC (Ab-120) Antibody:

• Single-cell epigenomics:

  • Adapt HIST1H2BC (Ab-120) Antibody for single-cell CUT&Tag or CUT&RUN

  • Map H2B variant distribution at single-cell resolution

  • Correlate with single-cell transcriptomics and chromatin accessibility

  • Identify cell type-specific roles of H2B variants in heterogeneous populations

• CRISPR-based epigenome editing:

  • Use CRISPR-dCas9 fused to histone-modifying enzymes to manipulate H2BK120 modifications

  • Validate targeting with HIST1H2BC (Ab-120) Antibody

  • Assess functional consequences on gene expression and chromatin organization

  • Create site-specific H2B variant replacements

• Spatial transcriptomics integration:

  • Combine HIST1H2BC (Ab-120) Antibody immunofluorescence with spatial transcriptomics

  • Correlate H2B variant distribution with spatially resolved gene expression

  • Map epigenetic territories within tissues and organoids

  • Identify spatial relationships between H2B variants and cellular phenotypes