HIST1H2BC (Ab-20) Antibody

What is HIST1H2BC and what is its role in cellular function?

HIST1H2BC is a core component of nucleosomes, the fundamental units of chromatin. As part of the histone H2B family, it plays a critical role in DNA packaging and gene regulation. HIST1H2BC is involved in chromatin structure maintenance, which directly impacts gene expression, DNA repair, and replication processes .

The protein is particularly important in maintaining proper chromosome organization, and its modifications (such as acetylation, ubiquitination, and 2-hydroxyisobutyrylation) are key regulators of chromatin accessibility .

What are the technical specifications of the HIST1H2BC (Ab-20) Antibody?

The HIST1H2BC (Ab-20) Antibody (PACO59665) is a rabbit polyclonal antibody specifically designed for detecting the HIST1H2BC protein. Key specifications include:

This antibody is designed to recognize the region surrounding lysine 20 (K20) of HIST1H2BC, which is a site known to undergo post-translational modifications .

How does the HIST1H2BC (Ab-20) antibody differ from other H2B antibodies?

The HIST1H2BC (Ab-20) antibody specifically targets the region around lysine 20 (K20) of human Histone H2B type 1-C/E/F/G/I. This specificity is important because:

• K20 acetylation (H2BK20ac) represents a distinct post-translational modification that can be used to distinguish active enhancers from other candidate cis-regulatory elements .

• Unlike antibodies targeting other regions such as H2BK5ac (EP857Y or D5H1S), H2BK11ac, or H2BK108 2-hydroxyisobutyrylation, the Ab-20 antibody provides specific information about the K20 modification state .

• The difference in target epitope allows researchers to investigate distinct regulatory pathways, as different lysine modifications on H2B correlate with varying transcriptional outcomes .

It's worth noting that antibody specificity should be validated experimentally, as cross-reactivity can occur between histone modification sites due to sequence similarities .

What are the validated applications for HIST1H2BC (Ab-20) Antibody?

The HIST1H2BC (Ab-20) Antibody has been validated for the following applications:

• Western Blot (WB): Successfully detects HIST1H2BC in whole cell lysates from various cell lines including 293 cells. The recommended dilution range is 1:100-1:1000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Can be used at dilutions of 1:2000-1:10000 for detection of HIST1H2BC in solution-phase samples .

Unlike some other HIST1H2BC antibodies (such as Ab-16), the Ab-20 variant has not been extensively validated for immunohistochemistry (IHC) or immunofluorescence (IF) applications according to the available data .

What are the recommended protocols for Western blot using HIST1H2BC (Ab-20) Antibody?

Based on optimized protocols for histone antibodies, the following Western blot procedure is recommended:

• Sample Preparation:

  • Lyse cells with RIPA buffer followed by brief sonication

  • Determine protein concentration by BCA assay

  • Mix with loading buffer and boil at 100°C for 5 minutes

  • Use 10-15 μg of total protein per lane

• Electrophoresis and Transfer:

  • Separate proteins on 15% SDS-PAGE gel (optimal for low molecular weight histones)

  • Transfer to PVDF membrane at 100V for 1 hour in cold transfer buffer

• Antibody Incubation:

  • Block membrane with Odyssey Blocking Buffer for 1.5 hours at room temperature

  • Incubate with HIST1H2BC (Ab-20) Antibody at 1:500 dilution overnight at 4°C

  • Wash 3× with TBST

  • Incubate with appropriate secondary antibody (anti-rabbit IgG) for 1 hour at room temperature

• Detection:

  • Visualize using chemiluminescence or fluorescence-based detection systems

  • Expected band size for HIST1H2BC: 14 kDa

• Controls:

  • Use beta-actin (approximately 42 kDa) or tubulin (approximately 55 kDa) as loading controls

  • Include a positive control sample (293 whole cell lysate is recommended)

How can researchers troubleshoot weak or non-specific signals?

When encountering weak or non-specific signals with HIST1H2BC (Ab-20) Antibody, consider the following troubleshooting steps:

• For weak signals:

  • Increase antibody concentration (try 1:100 dilution)

  • Extend primary antibody incubation time to 24-48 hours at 4°C

  • Enhance signal using more sensitive detection reagents

  • Increase protein loading (up to 20-25 μg for whole cell lysates)

  • Optimize extraction methods for better histone recovery

• For non-specific bands:

  • Increase blocking time or concentration (5% milk or BSA)

  • Perform additional washes with higher stringency (0.1-0.3% Tween-20)

  • Reduce antibody concentration (try 1:1000 dilution)

  • Use freshly prepared samples to minimize degradation products

  • Pre-adsorb antibody with non-specific proteins

• Cross-reactivity issues:

  • Be aware that high sequence similarity exists between H2BK5 and H3K27, and between H2BK20 and H2BK120, which may lead to cross-reactivity

  • Include peptide competition assays to confirm specificity

  • Compare results with other H2B antibodies targeting different epitopes

• Background reduction:

  • Filter buffers before use

  • Use freshly prepared reagents

  • Ensure thorough washing between incubation steps

How can HIST1H2BC (Ab-20) Antibody be used to study chromatin modifications?

The HIST1H2BC (Ab-20) Antibody provides valuable tools for investigating H2B modifications in chromatin research:

• Chromatin Immunoprecipitation (ChIP) Assays:

  • This antibody can be used to identify genomic regions where HIST1H2BC with K20 modifications are enriched

  • Protocols should be optimized for histone modifications, using crosslinking times of 10-15 minutes and sonication conditions that yield 200-500 bp DNA fragments

  • ChIP-seq analysis can reveal genome-wide distribution patterns of modified HIST1H2BC

• Co-Immunoprecipitation (Co-IP):

  • Can be used to identify protein complexes that interact with K20-modified HIST1H2BC

  • Particularly useful for studying enzymes that add or remove this modification

• Analysis of Enhancer Activity:

  • H2B modifications, particularly acetylation at K20, distinctively mark active enhancers

  • Can be used alongside H3K27ac antibodies to differentiate active from poised enhancers

  • Data shows that H2BNTac sites (including K20) can discriminate active enhancers from other candidate cis-regulatory elements

• Investigating Transcriptional Regulation:

  • Can be coupled with RNA-seq to correlate HIST1H2BC K20 modification with gene expression levels

  • Research shows that H2B modifications correlate with transcription activation and elongation

What is the relationship between HIST1H2BC modifications and disease states?

Research has identified several connections between HIST1H2BC modifications and disease:

• Cancer Biology:

  • Altered expression of HIST1H2BC and related variants has been observed in endocrine-resistant breast cancer

  • Studies show that histone H2B variants, including HIST1H2BC, are hypomethylated in estrogen deprivation-resistant cells compared to parental MCF-7 cells

  • Both overexpression and downregulation of H2B variants can affect cell proliferation, suggesting the need for tightly controlled expression

• Metabolic Regulation:

  • H2B degradation plays essential roles in gene expression under reduced insulin/IGF signaling

  • Blocking H2B degradation in daf-2 mutant animals (insulin/IGF receptor mutants) leads to failure in dauer formation and larval lethality

  • This suggests a critical role for H2B regulation in metabolic adaptation

• Epigenetic Dysregulation:

  • Modifications at specific lysine residues (including K20) can affect transcriptional programs

  • Aberrant histone modifications may contribute to various pathological conditions by altering gene expression profiles

  • Research has linked mutations in histone modification enzymes to Alzheimer's disease and congenital heart disease

How does HIST1H2BC ubiquitination and acetylation affect gene regulation?

Research has revealed important functional consequences of HIST1H2BC modifications:

• Ubiquitination:

  • H2B ubiquitylation at K123 (corresponding to human K120) is linked to gene activation

  • Studies show that mutation of this ubiquitylation site reduces expression of specific genes, such as GAL1 (reduced 4-fold) and SUC2 (reduced to 40% of wild-type levels)

  • The Rad6/Bre1 complex, which mediates this ubiquitylation, localizes at transcribed genes, suggesting a role in transcription elongation

  • Deubiquitylation by the SAGA complex component Ubp8 is also important for proper gene regulation

• Acetylation:

  • H2B N-terminal acetylation (H2BNTac), including at K20, distinctively marks active enhancers

  • These modifications strongly correlate with each other (Spearman's ρ = 0.84-0.93) but show modest correlation with H3K27ac, H3K9ac, and MED1

  • CBP/p300 catalytic activity is involved in H2BNTac, as demonstrated by reduced H2BNTac upon treatment with the CBP/p300 inhibitor A-485

  • H2BNTac is deacetylated by histone deacetylases (HDACs) 1 and 2

• 2-Hydroxyisobutyrylation:

  • A relatively newly discovered modification at K108 of HIST1H2BC

  • Likely plays a role in regulating chromatin structure and gene expression

  • Specialized antibodies have been developed to study this modification specifically

How does HIST1H2BC (Ab-20) Antibody compare with other histone H2B antibodies?

When selecting between different H2B antibodies, researchers should consider:

• Epitope Specificity:

  • HIST1H2BC (Ab-20) targets the region around K20

  • HIST1H2BC (Ab-16) targets the region around K16

  • Other antibodies target modifications such as K5, K11, or K108

  • Each provides information about different modification states and potentially different regulatory mechanisms

• Validated Applications:

  • Ab-20 is validated for WB and ELISA

  • Ab-16 is validated for WB, IHC, and IF

  • Other antibodies may have different application profiles

• Cross-Reactivity Profiles:

  • H2BK5ac EP857Y shows stronger correlation with H3K27ac compared to other H2BNTac antibodies

  • Sequence similarity between H2BK5 and H3K27, and between H2BK20 and H2BK120, can lead to cross-reactivity

  • Each antibody should be validated for specificity in the experimental system being used

• Species Reactivity:

  • Ab-20 reacts with human and mouse samples

  • Ab-16 reacts with human and rat samples

  • Consider species compatibility when designing experiments

What controls should be included when using HIST1H2BC (Ab-20) Antibody?

Proper experimental controls are essential when working with histone antibodies:

• Positive Controls:

  • 293 whole cell lysate (validated for Ab-20)

  • Other recommended cell lines include HeLa, A549, and HepG2 (shown to express detectable levels of HIST1H2BC)

• Negative Controls:

  • Secondary antibody only control (omit primary antibody)

  • Isotype control (rabbit IgG at equivalent concentration)

  • Cell lines with HIST1H2BC knockdown (using shRNA or CRISPR)

• Specificity Controls:

  • Peptide competition assay using the immunizing peptide

  • Comparison with other H2B antibodies targeting different epitopes

  • Testing antibody reactivity against purified histones or recombinant HIST1H2BC

• Loading Controls for Western Blot:

  • Beta-actin (approximately 42 kDa)

  • Tubulin (approximately 55 kDa)

  • For histone normalization, total H4 can be used (all 16 C. elegans H4 isoforms share identical protein sequence)

How can researchers validate HIST1H2BC (Ab-20) Antibody specificity for their experimental systems?

To confirm antibody specificity in your experimental system:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • A specific signal should be significantly reduced or eliminated

• Knockdown/Knockout Validation:

  • Generate HIST1H2BC knockdown cell lines using shRNA or CRISPR-Cas9

  • Compare antibody signal between control and knockdown samples

  • A specific antibody will show reduced signal in knockdown cells

• Mass Spectrometry Validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm that the precipitated protein is indeed HIST1H2BC

  • This approach can also identify post-translational modifications and interacting proteins

• Cross-Reactivity Testing:

  • Test against purified histones or histone peptides with similar sequences

  • Particularly important given the sequence similarities between H2BK20 and H2BK120

• Comparison with Other Antibodies:

  • Compare signals with other antibodies targeting different epitopes of HIST1H2BC

  • Consistent patterns suggest specificity, while discrepancies may indicate cross-reactivity issues

How can HIST1H2BC (Ab-20) Antibody be used in epigenetic research?

This antibody provides valuable tools for several cutting-edge epigenetic research applications:

• Chromatin State Mapping:

  • Use in ChIP-seq to map the genome-wide distribution of K20-modified HIST1H2BC

  • Integrate with other histone modification data to define chromatin states

  • Research shows that H2BNTac sites distinctively mark active enhancers and can be used to discriminate them from other candidate cis-regulatory elements

• Multi-Omics Integration:

  • Combine ChIP-seq data with RNA-seq to correlate HIST1H2BC modifications with gene expression

  • Integrate with ATAC-seq data to study the relationship between HIST1H2BC modifications and chromatin accessibility

  • Data shows that 71-92% of H3K27ac and H2BNTac peaks occur in ATAC-accessible regions

• Enzyme Function Studies:

  • Investigate the effects of histone modifying enzymes on HIST1H2BC

  • For example, CBP/p300 inhibition with A-485 reduces H2BNTac, while depletion of HDACs 1 and 2 increases it

• Developmental Biology:

  • Study changes in HIST1H2BC modifications during differentiation or development

  • H2B regulation has been shown to be critical for animal development under conditions of reduced insulin/IGF receptor activity

What emerging techniques can be combined with HIST1H2BC (Ab-20) Antibody for advanced research?

Several cutting-edge techniques can enhance research using this antibody:

• CUT&RUN and CUT&Tag:

  • These techniques offer advantages over traditional ChIP by providing higher signal-to-noise ratios and requiring fewer cells

  • Can be adapted for HIST1H2BC (Ab-20) Antibody to map K20 modifications with greater precision

• Single-Cell Epigenomics:

  • Combining HIST1H2BC antibody with single-cell techniques can reveal cell-to-cell variation in histone modifications

  • Particularly valuable for heterogeneous samples such as tumor tissues

• Live Cell Imaging:

  • Using derived intrabodies or nanobodies based on HIST1H2BC (Ab-20) for tracking dynamics of K20 modifications in living cells

• Proximity Ligation Assay (PLA):

  • Can detect interactions between HIST1H2BC and other proteins in situ

  • Useful for studying protein complexes involved in adding or removing K20 modifications

• CRISPR Screens Combined with Antibody-Based Readouts:

  • Use CRISPR libraries to identify genes affecting HIST1H2BC K20 modification

  • Screen for enzymes or regulatory factors using the antibody as a readout

How do HIST1H2BC modifications integrate with other histone marks in the histone code?

Research has revealed important relationships between HIST1H2BC modifications and other histone marks:

• Correlation with Other Histone Modifications:

  • H2BNTac sites (including K20ac) extensively overlap with each other (84-96%)

  • Most strongly marked H2BNTac regions are also marked with H3K27ac

  • H2BK5ac, H2BK12ac, H2BK16ac, and H2BK20ac strongly correlate with each other (Spearman's ρ = 0.84-0.93) but show modest correlation with H3K27ac, H3K9ac, and MED1

• Sequential Modification Mechanisms:

  • H2B ubiquitylation and subsequent deubiquitylation form a sequential mechanism for transcriptional activation

  • The SAGA complex component Ubp8 is required for SAGA-mediated deubiquitylation of H2B in vitro

• Coordinated Regulation:

  • H2B degradation affects DAF-16 binding to chromatin

  • This regulation is critical for insulin/IGF signaling and animal development

  • Preventing H2B degradation affects gene expression programs and can lead to developmental failure

• Cross-talk Between Different Histone Types:

  • H2B ubiquitination at K123 (human K120) is required for H3K4 and H3K79 methylation

  • This demonstrates how modifications on one histone can affect modifications on another histone, creating a complex regulatory network

This integrated network of histone modifications forms the basis of the histone code, which regulates gene expression in response to cellular and environmental signals.