HIST1H2BC (Ab-120) Antibody

What is HIST1H2BC (Ab-120) Antibody and what does it target?

HIST1H2BC (Ab-120) Antibody is a rabbit polyclonal antibody that specifically recognizes and binds to the human Histone H2B type 1-C/E/F/G/I protein. It targets a peptide sequence around the site of Lysine (120) derived from Human Histone H2B type 1-C/E/F/G/I. This antibody is critical for researchers investigating chromatin structure, histone modifications, and epigenetic regulation. The antibody has been validated for multiple applications including ELISA, Western Blot, and Immunohistochemistry, making it versatile for various experimental designs .

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

The HIST1H2BC (Ab-120) Antibody has been validated for several key research applications:

For IHC applications, the antibody has been specifically tested on paraffin-embedded human tissue samples using standard antigen retrieval protocols. The antibody consistently produces clear nuclear staining patterns in human tissues, reflecting the natural distribution of histone proteins .

What species reactivity does this antibody exhibit?

The HIST1H2BC (Ab-120) Antibody has confirmed reactivity with:

• Human (Homo sapiens) - Primary reactivity

• Rat (Rattus norvegicus) - Cross-reactivity

The antibody has been specifically designed against human protein sequences but shows cross-reactivity with rat samples due to the high conservation of histone proteins across mammalian species. This makes it valuable for comparative studies across these species .

How does post-translational modification of Lysine 120 affect antibody binding?

The HIST1H2BC (Ab-120) Antibody targets the region around Lysine 120 (K120) in Histone H2B type 1-C/E/F/G/I. Lysine 120 is a critical site for post-translational modifications (PTMs), particularly ubiquitination and acetylation, which play important roles in transcriptional regulation.

Researchers should note that PTMs at or near K120 may interfere with antibody binding. Specifically:

• Ubiquitination of K120 typically blocks antibody recognition

• Acetylation may partially impair binding efficiency

• Methylation may alter epitope structure and reduce binding

For experiments examining PTM impacts on chromatin structure, control experiments with modified and unmodified peptides are strongly recommended to establish detection specificity and sensitivity thresholds .

What are the methodological considerations for using this antibody in ChIP-seq experiments?

While not explicitly validated for ChIP-seq, researchers employing HIST1H2BC (Ab-120) Antibody in chromatin immunoprecipitation followed by sequencing should consider:

• Cross-linking protocol optimization: Use 1% formaldehyde for 10 minutes at room temperature as a starting point, followed by quenching with 125mM glycine.

• Sonication parameters: Adjust to achieve chromatin fragments of 200-500bp for optimal immunoprecipitation.

• Antibody concentration: Start with 3-5μg of antibody per ChIP reaction with 25-50μg of chromatin.

• Validation controls:

  • Include Input DNA control

  • Use IgG negative control

  • Consider H3K4me3 positive control for protocol verification

• Downstream analysis: Compare peaks against known H2B distribution patterns and correlate with transcriptional activity datasets.

Given the role of H2B in chromatin organization, this antibody may yield valuable insights into regulatory regions when applied to ChIP-seq workflows, though additional optimization beyond standard IHC/WB protocols will be necessary .

How can HIST1H2BC (Ab-120) Antibody be used in studies of chromatin remodeling complexes?

When investigating interactions between Histone H2B and chromatin remodeling complexes, HIST1H2BC (Ab-120) Antibody can be employed in multiple experimental approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Use 2-5μg antibody per 500-1000μg of nuclear extract

  • Include appropriate detergents (0.1% NP-40 recommended) to maintain complex integrity

  • Validate interactions with reciprocal IP experiments

• Proximity ligation assays (PLA):

  • Use 1:50 dilution alongside antibodies against remodeling complex components

  • Optimize fixation to preserve nuclear architecture (4% PFA for 15 minutes)

  • Perform rigorous specificity controls

• Sequential ChIP (ChIP-reChIP):

  • First IP with anti-remodeling complex antibody

  • Second IP with HIST1H2BC antibody at 1:20 dilution

  • Analyze enrichment at genomic regions of interest

The antibody's specificity for the K120 region makes it particularly valuable for studies examining how ubiquitination at this residue affects recruitment of specific remodeling complexes and subsequent chromatin restructuring events .

What are the optimal storage and handling conditions for HIST1H2BC (Ab-120) Antibody?

To maintain HIST1H2BC (Ab-120) Antibody performance and longevity, follow these storage and handling recommendations:

• Storage temperature: Store at -20°C or -80°C for long-term preservation.

• Working aliquots: Prepare small working aliquots (10-20μl) to avoid repeated freeze-thaw cycles. Each freeze-thaw cycle can reduce antibody activity by approximately 10%.

• Buffer composition: The antibody is supplied in 50% glycerol with 0.01M PBS (pH 7.4) and 0.03% Proclin 300 as a preservative. This formulation maintains stability during freeze-thaw cycles.

• Thawing procedure: Thaw at 4°C overnight or at room temperature for 30 minutes, followed by gentle mixing. Avoid vortexing, which can cause protein denaturation.

• Working dilutions: Prepare working dilutions immediately before use and discard any unused diluted antibody.

• Contamination prevention: Always use sterile technique when handling the antibody to prevent microbial contamination.

Following these guidelines will ensure optimal antibody performance and extend its useful life in experimental applications .

What protocol modifications are recommended for IHC applications with difficult tissue samples?

When working with challenging tissue samples for IHC applications of HIST1H2BC (Ab-120) Antibody, consider these protocol modifications:

• Antigen retrieval optimization:

  • For formalin-fixed tissues with extensive cross-linking, use high-pressure heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) at 120°C for 10 minutes

  • For tissues with high fat content, include a 0.1% Tween-20 in the retrieval buffer

  • For tissues with high background, try alternative retrieval methods such as enzymatic retrieval with proteinase K (10μg/ml for 10-15 minutes)

• Blocking enhancement:

  • Extend blocking time to 1-2 hours at room temperature

  • Use 5% BSA with 5% normal serum from the species of the secondary antibody

  • Consider adding 0.1% Triton X-100 to improve penetration

• Antibody incubation modifications:

  • Increase primary antibody concentration to 1:10 for difficult samples

  • Extend incubation to overnight at 4°C with gentle agitation

  • Add 0.05% Tween-20 to antibody diluent to reduce non-specific binding

• Signal amplification options:

  • Consider using polymer-based detection systems

  • Implement tyramide signal amplification for low-abundance targets

  • Use Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence in fluorescent applications

These modifications have proven successful for detecting HIST1H2BC in challenging samples such as archival tissues and samples with high melanin content .

How should Western blot protocols be optimized for HIST1H2BC detection?

For optimal detection of HIST1H2BC protein using Western blot analysis, implement these protocol refinements:

• Sample preparation:

  • Use histone extraction protocols with 0.2M H₂SO₄ or specialized histone extraction kits

  • Include protease inhibitors and deacetylase inhibitors (5mM sodium butyrate) in all buffers

  • Keep samples on ice throughout the procedure to prevent degradation

• Gel electrophoresis parameters:

  • Use 15-18% SDS-PAGE gels to effectively resolve the low molecular weight histone proteins (~14 kDa)

  • Add 1-5% SDS to the sample buffer to ensure complete denaturation

  • Consider using Triton-Acid-Urea (TAU) gels for separating histone variants with different modification states

• Transfer optimization:

  • Implement semi-dry transfer at 15V for 30 minutes

  • Use PVDF membranes (0.2μm pore size) pre-activated with methanol

  • Include 0.1% SDS in transfer buffer to facilitate protein movement

• Antibody incubation:

  • Block with 5% BSA in TBST for 2 hours at room temperature

  • Dilute primary antibody 1:500 to 1:2000 in 2% BSA in TBST

  • Incubate overnight at 4°C with gentle rocking

• Detection recommendations:

  • Use HRP-conjugated secondary antibodies at 1:5000 dilution

  • Develop using enhanced chemiluminescence (ECL) substrates

  • Exposure times may need adjustment due to varying expression levels of H2B variants

This optimized protocol addresses the specific challenges of working with histone proteins, which are small, highly basic, and often present in various modified forms .

What are the common causes of false negative results in HIST1H2BC (Ab-120) Antibody applications?

Researchers experiencing false negative results when using HIST1H2BC (Ab-120) Antibody should investigate these potential causes:

• Epitope masking:

  • Lysine 120 accessibility may be compromised by protein-protein interactions

  • Post-translational modifications (particularly ubiquitination) can obstruct antibody binding

  • Solution: Test alternative fixation methods or use epitope retrieval techniques

• Protein degradation:

  • Histones are susceptible to protease activity during sample preparation

  • Solution: Add protease inhibitors (PMSF, protease inhibitor cocktail) to all extraction buffers

• Technical issues:

  • Insufficient antigen retrieval (for IHC/IF)

  • Overfixation leading to excessive cross-linking

  • Solution: Optimize antigen retrieval time and method; reduce fixation time

• Sample type limitations:

  • The antibody is validated for human and rat samples

  • Cross-reactivity with other species may be limited or absent

  • Solution: Perform sequence alignment to determine epitope conservation in the species of interest

• Antibody deterioration:

  • Repeated freeze-thaw cycles

  • Improper storage conditions

  • Solution: Use fresh aliquots and store according to manufacturer recommendations

If troubleshooting basic protocol parameters does not resolve false negative results, consider validating antibody activity using a positive control sample with known HIST1H2BC expression .

How can background and non-specific binding be minimized in immunofluorescence applications?

To reduce background and non-specific binding when using HIST1H2BC (Ab-120) Antibody in immunofluorescence experiments:

• Blocking optimization:

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

  • Add 1% BSA to blocking buffer to reduce non-specific binding

  • Consider dual blocking with both serum and 0.5% casein for difficult samples

• Antibody dilution refinement:

  • Titrate antibody concentrations (start at 1:50 and test serial dilutions)

  • Prepare antibodies in blocking buffer rather than PBS alone

  • Filter antibody dilutions through a 0.22μm filter to remove aggregates

• Washing procedure enhancements:

  • Increase wash frequency (5-6 washes of 5 minutes each)

  • Use 0.1% Tween-20 or 0.1% Triton X-100 in wash buffers

  • Implement high-salt washes (500mM NaCl) for one washing step

• Autofluorescence reduction:

  • Treat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes

  • Use 10mM CuSO₄ in 50mM ammonium acetate buffer (pH 5.0) for 30 minutes

  • Photobleach samples with UV light for 15-30 minutes before antibody incubation

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Consider using F(ab')₂ fragments instead of whole IgG molecules

  • Pre-adsorb secondary antibodies with tissue powder from the sample species

Implementation of these techniques can significantly improve signal-to-noise ratio in immunofluorescence applications with HIST1H2BC (Ab-120) Antibody .

What strategies can resolve data inconsistencies between HIST1H2BC antibody results and other histone H2B detection methods?

When discrepancies arise between HIST1H2BC (Ab-120) Antibody results and other methods of histone H2B detection, consider these resolution approaches:

• Epitope-specific considerations:

  • The antibody targets the region around Lysine 120, which may be differentially accessible in various experimental contexts

  • Compare with antibodies targeting different H2B epitopes to determine if the discrepancy is epitope-specific

  • Use peptide competition assays to confirm specificity

• Histone variant distinction:

  • HIST1H2BC antibody recognizes multiple H2B variants (H2B type 1-C/E/F/G/I)

  • Discrepancies may arise when comparing to variant-specific detection methods

  • Use mass spectrometry to identify the exact H2B variants present in your samples

• Method-specific validation:

  • For ChIP experiments: Compare with ChIP-seq data using antibodies against different histone marks

  • For Western blots: Run 2D gels to separate variants and modifications

  • For IHC/IF: Perform dual staining with antibodies against different histone marks

• Cross-validation protocol:

  • Implement parallel detection with:

    • mRNA expression analysis (RT-qPCR)

    • Tagged H2B variant expression

    • Alternative commercial antibodies targeting the same protein

• Quantification standardization:

  • Use recombinant histone standards for absolute quantification

  • Normalize to total histone content rather than housekeeping proteins

  • Apply consistent image analysis parameters across all comparative methods

By systematically addressing these factors, researchers can resolve inconsistencies and gain deeper insights into histone biology and chromatin structure dynamics .

How can HIST1H2BC (Ab-120) Antibody contribute to cancer research?

HIST1H2BC (Ab-120) Antibody offers significant value for cancer research through multiple applications:

• Biomarker identification:

  • Altered H2B distribution and modification patterns correlate with various cancer types

  • The antibody can be used to assess H2B status in tissue microarrays and patient samples

  • Changes in HIST1H2BC expression have been observed in breast cancer tissue samples

• Epigenetic therapy response monitoring:

  • H2B modifications change in response to HDAC inhibitors and other epigenetic drugs

  • The antibody can track treatment-induced changes in H2B status

  • Correlation of H2B patterns with treatment outcomes may identify responder populations

• Chromatin accessibility studies:

  • Cancer progression involves significant changes in chromatin structure

  • HIST1H2BC antibody can be used in combination with DNase sensitivity assays

  • Results can reveal regions of altered chromatin compaction in malignant cells

• Cell differentiation and dedifferentiation:

  • H2B modification patterns shift during cellular differentiation and cancer-associated dedifferentiation

  • The antibody can monitor these changes in model systems and clinical samples

  • Data can provide insights into mechanisms of malignant transformation

Importantly, when used in human breast cancer tissue IHC, the antibody has demonstrated distinct nuclear staining patterns that differ between normal and malignant tissues, suggesting potential diagnostic applications .

What is the role of HIST1H2BC in genome engineering and gene editing contexts?

In genome engineering and gene editing research, HIST1H2BC (Ab-120) Antibody serves as a valuable tool for understanding chromatin-related factors affecting editing efficiency:

• Chromatin accessibility assessment:

  • CRISPR-Cas9 and other editing systems are influenced by chromatin state

  • The antibody can be used to map H2B distribution at target loci

  • Correlation between H2B status and editing efficiency provides insights for optimization

• DNA repair pathway investigation:

  • H2B modifications play roles in DNA damage response and repair pathway choice

  • The antibody can monitor H2B dynamics following editing-induced double-strand breaks

  • Data informs strategies to bias repair toward desired outcomes (HDR vs. NHEJ)

• Cell selection applications:

  • As mentioned in search result , toxin-based selection can be used for precise genome engineering

  • The antibody can verify chromatin states in successfully edited cells

  • Comparing pre- and post-selection chromatin structure helps understand selection mechanisms

• Integration site analysis:

  • H2B distribution influences transgene integration patterns

  • The antibody can map H2B status at integration hotspots

  • Results help predict integration sites and design better targeting strategies

These applications are particularly relevant for therapeutic cell engineering approaches, including CAR-T cell development and gene therapy vector design, where understanding and controlling the chromatin environment is critical for success .

How does HIST1H2BC (Ab-120) Antibody contribute to understanding histone dynamics during cell cycle progression?

The HIST1H2BC (Ab-120) Antibody provides crucial insights into histone dynamics throughout the cell cycle:

• Replication-dependent incorporation:

  • HIST1H2BC is a replication-dependent histone variant

  • The antibody can track new H2B incorporation during S-phase

  • When combined with EdU labeling, it reveals the timing and patterns of deposition

• Mitotic chromosome analysis:

  • H2B organization changes dramatically during mitotic chromosome condensation

  • The antibody can visualize H2B distribution in chromosomes at different mitotic stages

  • Analysis reveals mechanisms of chromosome structure maintenance

• Inheritance patterns:

  • During cell division, modified histones are distributed to daughter cells

  • The antibody can be used in pulse-chase experiments to track old vs. new H2B

  • Results illuminate epigenetic inheritance mechanisms

• Cell cycle checkpoint relationships:

  • H2B modifications are involved in checkpoint signaling

  • The antibody can monitor H2B status at cell cycle arrest points

  • Changes correlate with checkpoint activation and resolution

For optimal results in cell cycle studies, researchers should combine HIST1H2BC antibody staining with cell cycle markers (such as PCNA for S-phase or phospho-histone H3 for mitosis) in multi-parameter flow cytometry or immunofluorescence experiments. This approach enables precise correlation between cell cycle phase and H2B status at the single-cell level .