HIST1H2BC (Ab-20) Antibody

Basic Research Questions

• What is HIST1H2BC and what is its functional role in chromatin biology?

  HIST1H2BC (Histone Cluster 1 H2B Family Member C) is a core component of nucleosomes that plays a critical role in chromatin structure and gene regulation. As part of the histone family, it contributes to the packaging of DNA into chromatin, limiting DNA accessibility to cellular machineries. HIST1H2BC functions within the nucleosome to regulate transcription, DNA repair, DNA replication, and chromosomal stability .

  The protein contains several key domains and post-translational modification sites, including lysine 20 (K20), which can undergo acetylation and is the target of the Ab-20 antibody. These modifications are part of the "histone code" that regulates DNA accessibility and gene expression .

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

  Based on extensive validation studies, HIST1H2BC (Ab-20) Antibody has been successfully employed in multiple experimental applications:

  Application	Validation Status	Recommended Dilution
  Western Blot (WB)	Validated	1:100-1:1000
  ELISA	Validated	1:2000-1:10000
  Immunohistochemistry (IHC)	Validated	1:10-1:100
  Immunofluorescence (IF)	Validated	1:1-1:10
  Chromatin Immunoprecipitation (ChIP)	Validated in some formulations	Varies by formulation

  The antibody has demonstrated consistent results in detecting the native protein in cellular lysates and tissue samples, with particular efficacy in human breast cancer and brain tissue sections .

• How do different HIST1H2BC antibody clones compare in their epitope recognition and applications?

  Different antibody clones targeting HIST1H2BC recognize distinct epitopes and demonstrate varying utility across applications:

  Antibody Identifier	Clone Type	Epitope Region	Key Applications	Host Species
  PACO59665 (Ab-20)	Polyclonal	Lys20 region	ELISA, WB	Rabbit
  PACO60514 (Ab-20)	Polyclonal	Lys20 region	ELISA, IHC, IF	Rabbit
  PACO60504 (Ab-16)	Polyclonal	Lys16 region	ELISA, WB, IHC, IF	Rabbit
  AMAb91337	Monoclonal	Proprietary	IHC, WB	Mouse

  The Ab-20 antibodies specifically target the region surrounding lysine 20, which is a critical site for post-translational modifications such as acetylation. This specificity makes them particularly valuable for studying histone modifications in chromatin regulation research .

Advanced Research Applications

• How can I optimize ChIP protocols using HIST1H2BC (Ab-20) Antibody for epigenetic studies?

  Optimizing ChIP protocols with HIST1H2BC (Ab-20) Antibody requires several key considerations:

  1. Crosslinking optimization: For histone proteins, use 1% formaldehyde for 10 minutes at room temperature; excessive crosslinking can mask epitopes.

  2. Sonication parameters: Aim for chromatin fragments of 200-500bp for optimal resolution.

  3. Antibody concentration: Begin with 2-5 μg of antibody per ChIP reaction; titration may be necessary.

  4. Buffer conditions: Use a buffer containing 50% glycerol and 0.01M PBS (pH 7.4) to maintain antibody stability.

  5. Controls: Include IgG negative control and a positive control targeting a known abundant histone mark.

  Research has shown that HIST1H2BC enrichment patterns in ChIP-seq experiments often correlate with H2B ubiquitylation marks, particularly in transcriptionally active regions . When analyzing ChIP-seq data, compare enrichment patterns with H3K4me3 marks, as H2B modifications can influence H3 methylation states .

• What is the relationship between H2B ubiquitylation and HIST1H2BC function in transcriptional regulation?

  H2B ubiquitylation plays a complex, context-dependent role in transcriptional regulation that researchers should consider when using HIST1H2BC antibodies:

  1. Co-transcriptional association: The majority of H2B ubiquitylation (H2Bub1) in chromatin localizes to gene coding regions through association of the Bre1 ubiquitin ligase and Rad6 ubiquitin conjugase with elongating RNA Polymerase II .

  2. Dynamic regulation: Cycles of ubiquitylation and deubiquitylation during transcription are prerequisites for optimal gene expression .

  3. Dual effects on transcription:

     • At promoters: H2Bub1 can repress expression by opposing recruitment of chromatin remodelers like RSC .

     • In gene bodies: H2Bub1 can facilitate transcription elongation .

  4. Selective gene regulation: Only a subset of genes is transcriptionally affected by loss of H2B ubiquitylation, including histones H2A/H2B and p53, while RNF20 (human Bre1) suppresses expression of several proto-oncogenes .

  When using HIST1H2BC (Ab-20) antibody in transcriptional studies, researchers should consider these dual roles and coordinate with antibodies recognizing ubiquitylated H2B for comprehensive analysis .

• How can the HIST1H2BC (Ab-20) Antibody be utilized to study DNA replication mechanisms?

  HIST1H2BC (Ab-20) Antibody can be instrumental in investigating the role of histone H2B in DNA replication:

  1. Chromatin association studies:

     • ChIP-qPCR targeting replication origins shows H2Bub1 is present in chromatin adjacent to origins of replication .

     • Combine with antibodies against pre-replication complex components (e.g., MCM proteins) to study temporal dynamics.

  2. Replication stress response:

     • H2B ubiquitylation is important for the cellular response to replication stress, with htb-K123R mutants showing sensitivity to hydroxyurea .

     • Use HIST1H2BC (Ab-20) Antibody in conjunction with phospho-specific antibodies against checkpoint kinase targets to analyze replication stress signaling.

  3. Replication fork progression:

     • In the absence of H2B ubiquitylation, there is decreased association of factors required for DNA synthesis and impaired progression of replication forks .

     • Combine antibody with DNA fiber analysis to examine replication fork dynamics.

  4. Nucleosome assembly on replicated DNA:

     • Histone occupancy is impaired around replicated origins in the absence of H2Bub1 .

     • Use HIST1H2BC (Ab-20) Antibody to track histone deposition on newly synthesized DNA.

  Experimental design should account for cell cycle synchronization, with appropriate controls for different cell cycle phases when analyzing HIST1H2BC dynamics during replication .

• What are the key considerations for immunofluorescence applications using HIST1H2BC (Ab-20) Antibody?

  When performing immunofluorescence with HIST1H2BC (Ab-20) Antibody, researchers should address several critical parameters:

  1. Fixation and permeabilization:

     • Recommended protocol: 4% formaldehyde fixation for 10-15 minutes

     • Permeabilize with 0.2% Triton X-100 for nuclear antigen access

  2. Blocking conditions:

     • Use 10% normal goat serum for 30 minutes at room temperature to minimize non-specific binding

  3. Antibody dilution and incubation:

     • Optimal dilution: 1:1 to 1:10 for most cell types

     • Incubate overnight at 4°C for maximum signal-to-noise ratio

  4. Secondary antibody selection:

     • Anti-rabbit IgG conjugated with Alexa Fluor 488 works effectively with this primary antibody

     • Counter-staining with DAPI helps visualize nuclei and assess H2B distribution

  5. Controls and validation:

     • Include peptide competition controls to verify specificity

     • H2B is ubiquitously expressed, so all nuclei should show positive staining

     • The pattern should be nuclear with potential heterogeneity reflecting chromatin states

  Published immunofluorescence studies using this antibody have successfully visualized HIST1H2BC in HepG2 cells, showing distinct nuclear localization patterns that correlate with chromatin condensation states .

Technical Considerations and Troubleshooting

• How can I distinguish between HIST1H2BC and other H2B variants in my experimental system?

  Distinguishing between H2B variants requires careful experimental design due to high sequence similarity:

  1. Antibody selection strategies:

     • The HIST1H2BC (Ab-20) Antibody specifically targets the region around lysine 20, which can differ between variants

     • Verify specificity through peptide competition assays with variant-specific peptides

  2. Western blot considerations:

     • Use high-resolution gels (15-20% polyacrylamide) to separate closely related variants

     • Include positive controls with recombinant proteins of specific variants

     • Sequential probing with antibodies targeting different H2B variants can reveal expression patterns

  3. Mass spectrometry approach:

     • For definitive identification, use LC-MS/MS analysis of immunoprecipitated samples

     • Focus on variant-specific peptides, particularly from N-terminal tails which show greater sequence divergence

  4. Variant-specific expression patterns:

     • Some H2B variants show tissue-specific expression; for example, H2B.W1 is testis-specific

     • Different H2B variants may show different temporal expression during development or disease progression

  Research has shown that altered ratios of H2B variants (such as H2B.1/H2B.2) may correlate with malignant progression in certain cancers, making accurate distinction between variants clinically relevant .

• What is the impact of post-translational modifications on HIST1H2BC (Ab-20) antibody recognition?

  Post-translational modifications (PTMs) can significantly affect epitope recognition by the HIST1H2BC (Ab-20) Antibody:

  1. Lysine 20 modifications:

     • The Ab-20 antibody targets the region around lysine 20 (K20)

     • K20 can undergo acetylation (K20ac), which is recognized by specific antibodies like ab240890 and ab177430

     • If K20 is acetylated, the Ab-20 antibody may show reduced binding affinity

  2. Adjacent modifications:

     • Modifications at adjacent residues (like K16 or K23) may affect Ab-20 binding through steric hindrance or conformational changes

     • Studies have shown that histone modifications can exhibit "crosstalk" where one modification influences another

  3. Methodological considerations:

     • When studying specific modifications, use modification-specific antibodies (e.g., anti-H2BK20ac)

     • For general HIST1H2BC detection regardless of modification state, target regions less prone to PTMs

     • To study multiple modifications simultaneously, consider sequential ChIP (re-ChIP) approaches

  4. Functional implications:

     • H2B ubiquitylation at K120/K123 (yeast) affects transcription and replication

     • ADP-ribosylation of H2B occurs at glutamate/aspartate residues with PARP-1/NMNAT-1, but at serine residues with PARP-1/HPF1

  When investigating specific PTMs, researchers should use antibodies specifically validated for the modified epitope, as general antibodies may show variable affinity depending on the modification status .

• What are the best experimental controls when using HIST1H2BC (Ab-20) Antibody in ChIP and immunoblotting studies?

  Rigorous experimental controls are essential for reliable results with HIST1H2BC (Ab-20) Antibody:

  For ChIP experiments:

  1. Input DNA control:

     • Reserve 5-10% of chromatin before immunoprecipitation

     • Essential for normalization and calculating percent input enrichment

  2. Negative controls:

     • IgG control from the same species as the primary antibody

     • Non-expressing regions (gene deserts) for background assessment

  3. Positive controls:

     • Housekeeping genes with known H2B enrichment

     • For histone studies, include regions with established histone profiles

  4. Peptide competition:

     • Pre-incubate antibody with excess target peptide to confirm specificity

     • Should abolish or significantly reduce specific signal

  For Western blot:

  1. Loading controls:

     • Total histone H3 or H4 for chromatin fraction normalization

     • Total protein staining (Ponceau S) for general loading control

  2. Positive controls:

     • Cell lines with known HIST1H2BC expression (HeLa, 293, A549, HepG2)

     • Recombinant HIST1H2BC protein standard curve for quantification

  3. Negative controls:

     • Lysates treated with histone extraction protocols vs. standard protocols

     • Peptide competition to confirm band specificity

  4. Validation across systems:

     • Compare results across multiple detection methods (e.g., different secondary antibodies, detection systems)

     • Test reproducibility in different cell types with known HIST1H2BC expression

  Published studies have validated HIST1H2BC (Ab-20) antibody in multiple cell lines including HeLa, 293, A549, and HepG2, with consistent detection of the expected 14 kDa band in western blot applications .

Research Applications in Disease Models

• How is HIST1H2BC antibody used to study the role of histone variants in cancer research?

  HIST1H2BC antibody has become an important tool in cancer research to investigate epigenetic dysregulation:

  1. Expression profiling in tumors:

     • IHC studies using HIST1H2BC antibodies have revealed altered expression patterns in various cancers

     • The antibody has been validated for paraffin-embedded human breast cancer tissue samples

     • Researchers can quantify expression levels through IHC scoring systems or western blot densitometry

  2. Epigenetic regulation in oncogenesis:

     • H2B ubiquitylation levels, detectable using specific antibodies, have tumor suppressor functions

     • RNF20 (the major H2B-specific E3 ubiquitin ligase) suppresses expression of proto-oncogenes, while its depletion can elicit transformation and tumorigenesis

     • H2B variant ratio changes (e.g., H2B.2:H2B.1) have been correlated with malignancy in Friend erythroleukemia

  3. Methodological approaches:

     • Tissue microarray analysis to screen multiple patient samples

     • ChIP-seq to map genome-wide H2B variant distribution in normal vs. cancer cells

     • Integration with transcriptomic data to identify correlation between H2B variant expression and gene expression patterns

  4. H2B variants as biomarkers:

     • Downregulation of H2B.1 has been observed in pediatric brain tumors

     • RNF20 promoter hypermethylation is frequently observed in tumors, suggesting altered H2B ubiquitylation

  When designing cancer studies using HIST1H2BC antibodies, researchers should include appropriate tissue-matched normal controls and consider cell type heterogeneity within tumor samples for accurate interpretation .

• What protocols are recommended for studying the dynamics of HIST1H2BC in cell cycle progression?

  To investigate HIST1H2BC dynamics during cell cycle progression, researchers should implement these methodological approaches:

  1. Cell synchronization techniques:

     • Double thymidine block for G1/S boundary synchronization

     • Nocodazole treatment for mitotic arrest

     • Serum starvation-release for G0/G1 synchronization

     • Hydroxyurea (HU) treatment for S-phase arrest

  2. Cell cycle monitoring:

     • Flow cytometry with propidium iodide for DNA content analysis

     • EdU incorporation to track S-phase cells

     • Cyclin B1/Clb2 immunoblotting as G2/M marker

     • H3K56ac as S-phase marker

  3. HIST1H2BC dynamics assessment:

     • Time-course ChIP analysis at replication origins and transcribed regions

     • Immunofluorescence microscopy combined with cell cycle markers

     • FRAP (Fluorescence Recovery After Photobleaching) with GFP-tagged H2B to study mobility

  4. Replication-specific protocols:

     • Nascent DNA immunoprecipitation (NasChIP) to study newly incorporated histones

     • DNA fiber analysis combined with immunofluorescence

     • iPOND (isolation of Proteins On Nascent DNA) coupled with HIST1H2BC antibody detection

  Research has demonstrated that H2B ubiquitylation plays important roles in S-phase progression, with htb-K123R mutants showing approximately 15-minute longer S-phase duration in unperturbed cell cycles . The HIST1H2BC (Ab-20) antibody can be used to track H2B dynamics throughout the cell cycle, revealing associations with replication origins in both G1 and G2/M phases .