HIST1H2BC (Ab-12) Antibody

What is HIST1H2BC and what role does it play in chromatin organization?

HIST1H2BC is a member of the histone H2B family involved in chromatin structure and gene regulation. It plays a critical role in nucleosome formation, which is the basic repeating unit of chromatin. As part of the histone octamer, HIST1H2BC contributes to DNA packaging and accessibility, directly impacting gene expression patterns . The protein serves as a scaffold around which DNA wraps, forming the first level of chromatin organization. Post-translational modifications of HIST1H2BC, including ubiquitylation and acetylation, regulate chromatin dynamics and influence fundamental cellular processes including transcription, DNA replication, and DNA repair mechanisms .

What applications is HIST1H2BC (Ab-12) Antibody validated for?

The HIST1H2BC (Ab-12) Antibody (PACO60494) has been validated for multiple experimental applications, providing researchers with versatility in experimental design:

The antibody has demonstrated positive Western blot detection in numerous cell lines including HeLa, 293, A549, K562, and HepG2 whole cell lysates, as well as in rat liver tissue, rat kidney tissue, mouse brain tissue, and mouse spleen tissue . This broad validation makes it suitable for diverse experimental systems exploring histone biology.

How should samples be prepared for optimal HIST1H2BC detection?

For optimal detection of HIST1H2BC, sample preparation should account for the nuclear localization of histones and their tight association with chromatin. Cell lysis should be performed using specialized buffers containing detergents capable of disrupting nuclear membranes (e.g., RIPA buffer supplemented with SDS). When preparing samples for Western blotting, it is crucial to include protease inhibitors to prevent degradation of histone proteins. Additionally, histone extraction protocols often benefit from acid extraction methods using 0.2N HCl or 0.4N H2SO4, which effectively separates histones from DNA.

For immunofluorescence applications, cells should be fixed with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100 to allow antibody access to nuclear proteins. For tissue sections in IHC, antigen retrieval steps (typically heat-induced epitope retrieval in citrate buffer pH 6.0) are essential to expose the HIST1H2BC epitope that may be masked during fixation processes .

What species reactivity does the HIST1H2BC (Ab-12) Antibody demonstrate?

The HIST1H2BC (Ab-12) Antibody exhibits broad species cross-reactivity, making it valuable for comparative studies across different model organisms:

This cross-species reactivity is due to the high conservation of histone proteins across mammalian species. The antibody was generated using an immunogen consisting of a peptide sequence around site of Lys (12) derived from Human Histone H2B type 1-C/E/F/G/I . Researchers should still perform validation in their specific experimental systems, as epitope accessibility may vary across tissue types and fixation methods.

What are the storage and handling recommendations for HIST1H2BC (Ab-12) Antibody?

To maintain antibody integrity and performance, proper storage and handling of the HIST1H2BC (Ab-12) Antibody is essential. The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative . For optimal stability:

• Store the antibody at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by aliquoting the antibody into smaller volumes upon receipt

• When removing from storage, thaw the antibody slowly on ice

• Centrifuge briefly before opening the tube to ensure all liquid is at the bottom

• Return to -20°C immediately after use

• For working dilutions, use fresh buffer preparations to minimize contamination risk

When properly stored and handled, the antibody typically maintains reactivity for at least 12 months from the date of receipt.

How does H2B ubiquitylation impact DNA replication and what methodologies can detect this relationship?

H2B ubiquitylation (H2Bub1) plays a critical role in DNA replication through multiple mechanisms. Research has shown that H2Bub1 is present in chromatin adjacent to origins of replication in yeast and is maintained during replication via the association of Bre1 (the E3 ligase for H2B) with newly replicated DNA . Methodologically, these relationships can be investigated through:

• Chromatin Immunoprecipitation (ChIP): Using antibodies against H2Bub1 to map its presence at replication origins

• Sequential ChIP: To determine co-localization of H2Bub1 with replication factors

• Nascent DNA capture: To identify newly synthesized DNA associated with H2Bub1

Cells lacking H2Bub1 (using htb-K123R mutants in yeast) show increased sensitivity to hydroxyurea (HU), indicating a role in replication stress response. While the pre-replication complex (pre-RC) forms normally in these cells, there is decreased association of factors required for DNA synthesis, leading to defects in replication fork progression and replisome destabilization . Experimentally, this has been demonstrated through:

• FACS analysis showing delayed completion of DNA synthesis in htb-K123R cells (approximately 20 minutes longer than wild type) following HU release

• Monitoring of H3K56ac (S phase marker) and Clb2 (G2/M marker) to track cell cycle progression

• Replication timing assays showing prolonged S-phase (~15 minutes longer) even in unperturbed cell cycles

These methodologies provide robust approaches for researchers investigating H2B ubiquitylation in relation to DNA replication.

What are the technical considerations for using HIST1H2BC (Ab-12) Antibody in ChIP experiments?

When designing ChIP experiments using HIST1H2BC (Ab-12) Antibody, several technical aspects require careful optimization:

• Crosslinking conditions: For histone ChIP, a shorter crosslinking time (5-10 minutes with 1% formaldehyde) is typically optimal, as over-crosslinking can reduce epitope accessibility.

• Sonication parameters: Chromatin should be sheared to 200-500bp fragments. This typically requires optimization of sonication cycles and intensity for each cell type.

• Antibody amount: Use 2-5μg of HIST1H2BC (Ab-12) Antibody per ChIP reaction with 25-30μg of chromatin. Titration experiments are recommended to determine optimal antibody:chromatin ratios.

• Controls:

  • Input control (10% of pre-immunoprecipitation chromatin)

  • IgG negative control (using matched rabbit IgG)

  • Positive control regions (known H2B-enriched loci)

  • Negative control regions (H2B-depleted loci)

• Washing stringency: Balance between maintaining specific interactions while removing non-specific binding. Typically, use low-salt, high-salt, LiCl, and TE washing buffers in sequence.

• Elution and reversal of crosslinks: Complete elution typically requires 65°C incubation overnight with proteinase K treatment.

• Quantification method: qPCR for targeted loci or sequencing (ChIP-seq) for genome-wide analysis. For ChIP-seq, additional considerations regarding library preparation, sequencing depth, and bioinformatic analysis are necessary.

Optimization of these parameters will enhance the specificity and sensitivity of HIST1H2BC detection in ChIP experiments.

What methods can be used to validate HIST1H2BC (Ab-12) Antibody specificity?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For HIST1H2BC (Ab-12) Antibody, several complementary approaches can be employed:

• Peptide competition assays: Pre-incubating the antibody with excess immunizing peptide should abolish specific signal in applications like Western blot or immunofluorescence.

• Genetic validation:

  • Using HIST1H2BC knockout/knockdown cells as negative controls

  • Testing antibody reactivity in cells overexpressing tagged HIST1H2BC

• Mass spectrometry validation: Confirming the identity of immunoprecipitated proteins by mass spectrometry analysis.

• Peptide array analysis: Testing antibody reactivity against a panel of histone peptides representing various H2B variants and modifications to assess cross-reactivity profiles .

• Western blot analysis: Looking for a single band at the expected molecular weight (14 kDa) in different cell types. The antibody has been validated in HeLa, 293, A549, K562, and HepG2 whole cell lysates .

• Immunoprecipitation followed by Western blot: Using a different HIST1H2BC antibody recognizing a distinct epitope to confirm specificity.

The Histone Antibody Specificity Database (http://www.histoneantibodies.com) provides a valuable resource for comparing antibody performance and cross-reactivity assessed by peptide microarray analysis . This database can help researchers evaluate antibody behavior before designing experiments.

How do post-translational modifications affect HIST1H2BC function and antibody recognition?

Post-translational modifications (PTMs) of HIST1H2BC significantly impact its functional properties and can influence antibody recognition. Major PTMs of H2B include:

• Ubiquitylation: Particularly at lysine 120 (K120) in humans, which impacts transcriptional regulation and DNA repair

• Acetylation: At multiple lysine residues including K5, K12, K15, K20, and K120, generally associated with transcriptional activation

• Methylation: Less common but observed at several residues

• Phosphorylation: Particularly at serine 14 (S14), associated with apoptosis and DNA damage response

The HIST1H2BC (Ab-12) Antibody targets the region around lysine 12, which is a known acetylation site . This creates important technical considerations:

• Epitope masking: Acetylation at K12 may mask the epitope, potentially reducing antibody binding if the antibody recognizes the unmodified form.

• Modification specificity: The HIST1H2BC (Ab-12) Antibody documentation should be consulted to determine if it recognizes the modified or unmodified state of K12.

• Context-dependent recognition: Adjacent modifications can influence antibody accessibility to its target epitope.

For researchers specifically interested in acetylation at K12, specialized antibodies like the Acetyl-Histone H2B (Lys12) Antibody are available and have been validated for Western blotting, immunoprecipitation, and immunofluorescence applications . When studying PTMs, consider these methodological approaches:

• Use modification-specific antibodies for direct detection

• Employ mass spectrometry for comprehensive PTM mapping

• Utilize pharmacological inhibitors or activators of writer/eraser enzymes to manipulate modification states

• Implement site-directed mutagenesis to create modification-mimetic or modification-deficient mutants

Understanding the interplay between different PTMs and their impact on HIST1H2BC function requires careful experimental design and validation of antibody specificity in the context of various modifications.

What are common issues when using HIST1H2BC (Ab-12) Antibody in Western blotting and how can they be resolved?

When using HIST1H2BC (Ab-12) Antibody for Western blotting, researchers may encounter several technical challenges. Here are common issues and their solutions:

• Weak or no signal:

  • Increase antibody concentration (try 1:100 dilution instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Increase protein loading amount (20-30μg for whole cell lysates)

  • Use enhanced chemiluminescence (ECL) substrate with higher sensitivity

  • Ensure efficient transfer of low molecular weight proteins by using PVDF membrane with 0.2μm pore size

  • Include 0.1% SDS in transfer buffer to improve histone transfer

• High background:

  • Increase blocking time (2 hours at room temperature)

  • Use 5% BSA instead of milk for blocking and antibody dilutions

  • Increase washing duration and number of washes (5 x 5 minutes with TBST)

  • Decrease antibody concentration (try 1:1000 instead of 1:100)

  • Ensure membranes are completely covered during all incubation steps

• Multiple bands:

  • This may reflect cross-reactivity with other H2B variants due to sequence similarity

  • Use more stringent washing conditions

  • Pre-adsorb antibody with recombinant histone proteins of other variants

  • Consider using acid extraction to isolate histones, which can provide cleaner results

• Inconsistent results between experiments:

  • Standardize protein extraction protocol, especially for nuclear proteins

  • Use validated positive control lysates (e.g., HeLa or 293 cells)

  • Implement internal loading controls (total histone H3 or H4)

  • Prepare fresh working dilutions of antibody for each experiment

Implementing these troubleshooting strategies should improve the reliability and consistency of HIST1H2BC detection by Western blotting.

How can HIST1H2BC (Ab-12) Antibody be optimized for immunofluorescence applications?

Optimizing immunofluorescence (IF) protocols for HIST1H2BC (Ab-12) Antibody requires attention to several key parameters:

• Fixation method optimization:

  • Test both paraformaldehyde (PFA, 2-4%) and methanol fixation

  • For PFA fixation, compare 10 minutes at room temperature versus 20 minutes

  • For methanol fixation, use ice-cold methanol for 10 minutes at -20°C

  • In some cases, dual fixation (PFA followed by methanol) may improve nuclear protein detection

• Permeabilization optimization:

  • Test different concentrations of Triton X-100 (0.1-0.5%)

  • Compare Triton X-100 with other detergents (0.1-0.5% Tween-20 or 0.1% SDS)

  • Extend permeabilization time (10-30 minutes) for better nuclear access

• Antibody concentration:

  • Start with the recommended 1:1-1:10 dilution range

  • Prepare a dilution series (e.g., 1:1, 1:5, 1:10, 1:25) to determine optimal concentration

  • Extend primary antibody incubation to overnight at 4°C for improved signal

• Antigen retrieval methods:

  • Test heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Compare with Tris-EDTA buffer (pH 9.0)

  • Optimize heating time (10-20 minutes)

• Signal amplification:

  • Use tyramide signal amplification for weak signals

  • Try biotin-streptavidin amplification systems

  • Select secondary antibodies with bright fluorophores (e.g., Alexa Fluor 488 or 594)

• Controls and validation:

  • Include a no-primary antibody control

  • Use HIST1H2BC-depleted cells as negative controls

  • Compare staining pattern with other validated H2B antibodies

• Imaging optimization:

  • Adjust exposure settings to avoid saturation

  • Use deconvolution for improved resolution

  • Consider confocal microscopy for better subcellular localization

By systematically optimizing these parameters, researchers can achieve specific and robust HIST1H2BC detection in immunofluorescence applications.

How can HIST1H2BC (Ab-12) Antibody be used to investigate the relationship between histone modifications and DNA replication?

Investigating the relationship between HIST1H2BC modifications and DNA replication requires sophisticated experimental approaches that integrate chromatin analysis with replication tracking. The HIST1H2BC (Ab-12) Antibody can be employed in several advanced experimental strategies:

• Sequential ChIP (Re-ChIP): This technique can determine co-occurrence of HIST1H2BC and specific modifications at replication origins:

  • First ChIP with HIST1H2BC (Ab-12) Antibody

  • Elute chromatin complexes under mild conditions

  • Second ChIP with antibodies against specific modifications (e.g., ubiquitylation, acetylation)

  • qPCR analysis targeting known replication origins

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq) combined with replication analysis:

  • Perform HIST1H2BC ChIP-seq in synchronized cell populations at different stages of S phase

  • Integrate with nascent DNA sequencing techniques (e.g., Repli-seq, OK-seq)

  • Map HIST1H2BC enrichment patterns relative to replication timing domains

• Proximity Ligation Assay (PLA) to detect HIST1H2BC-replication protein interactions in situ:

  • Co-stain cells with HIST1H2BC (Ab-12) Antibody and antibodies against replication factors

  • Use species-specific PLA probes to detect close proximity (<40nm)

  • Quantify PLA signals during different stages of the cell cycle

• iPOND (isolation of Proteins On Nascent DNA) combined with HIST1H2BC detection:

  • Label newly synthesized DNA with EdU

  • Perform click chemistry to biotinylate EdU-labeled DNA

  • Isolate protein complexes on nascent DNA using streptavidin beads

  • Detect HIST1H2BC presence at replication forks by Western blotting

• FRAP (Fluorescence Recovery After Photobleaching) analysis of HIST1H2BC dynamics:

  • Generate cells expressing fluorescently tagged HIST1H2BC

  • Analyze recovery rates in the presence or absence of replication inhibitors

  • Compare dynamics at active replication foci versus non-replicating regions

Research has demonstrated that H2B ubiquitylation impacts DNA replication through effects on replisome stability and nucleosome assembly on newly replicated DNA . Similar methodologies can be applied to investigate other HIST1H2BC modifications and their influence on replication processes.

What is the current understanding of HIST1H2BC modifications in cancer biology and therapeutic resistance?

The role of HIST1H2BC and its modifications in cancer biology is an emerging area of research with significant implications for understanding therapeutic resistance. While specific studies on HIST1H2BC are still developing, related histone H2B variants like HIST1H2BK have been implicated in chemotherapy resistance:

• Modification-specific roles:

  • Ubiquitylation of H2B has been linked to DNA damage response and repair pathways, which can influence therapy resistance

  • Acetylation at K12 and other residues affects chromatin accessibility and gene expression patterns in cancer cells

  • Phosphorylation at S14 is associated with apoptotic pathways and may influence response to cytotoxic therapies

• Cancer-specific alterations:

  • Changes in HIST1H2BC expression levels have been observed in various cancer types

  • Alterations in enzymes that modify HIST1H2BC (writers, erasers, readers) are common in cancer and can affect therapy response

  • Cancer-specific mutations in histones (oncohistones) can disrupt normal modification patterns

• Therapeutic resistance mechanisms:

  • Related histone HIST1H2BK has been shown to inhibit 5-FU-induced apoptosis through activation of A2M transcription and LRP1/PI3K/Akt signaling

  • This suggests histone H2B variants may play separate but coordinated roles in therapeutic response

  • Specific HIST1H2BC modifications may similarly affect drug sensitivity through regulation of DNA repair or apoptotic pathways

Methodological approaches to investigate HIST1H2BC in cancer resistance include:

• Comparative proteomics of sensitive versus resistant cell lines to identify differential HIST1H2BC modifications

• CRISPR-based screens to assess the impact of HIST1H2BC depletion on drug sensitivity

• ChIP-seq to map HIST1H2BC occupancy changes in response to therapy

• Integration of HIST1H2BC modification data with transcriptomic profiles to identify regulated pathways

• Development of modification-specific inhibitors targeting enzymes that modify HIST1H2BC

As research progresses, a more complete understanding of HIST1H2BC modifications in cancer may reveal new biomarkers for therapy response prediction or novel therapeutic targets.

What emerging technologies will enhance HIST1H2BC research in the coming years?

Several cutting-edge technologies are poised to revolutionize HIST1H2BC research and expand our understanding of its functions:

• Single-cell epigenomics: Technologies like single-cell ChIP-seq, CUT&Tag, and CUT&RUN will enable mapping of HIST1H2BC distribution at unprecedented resolution, revealing cell-to-cell heterogeneity.

• Genome editing with base editors and prime editors: More precise than traditional CRISPR-Cas9, these tools will allow site-specific modification of HIST1H2BC to introduce or remove specific post-translational modification sites.

• Live-cell imaging of histone dynamics: Improvements in fluorescent probes and super-resolution microscopy will enable real-time tracking of HIST1H2BC behavior during cellular processes.

• Mass spectrometry advancements: Enhanced sensitivity in technologies like top-down proteomics will improve detection of HIST1H2BC modification patterns and combinatorial modifications.

• Cryo-electron microscopy (cryo-EM): Higher resolution structures of nucleosomes containing HIST1H2BC with specific modifications will provide mechanistic insights into how these modifications affect chromatin architecture.

• Microfluidics and organ-on-chip systems: These platforms will enable studies of HIST1H2BC dynamics in more physiologically relevant contexts and with greater throughput.

• Artificial intelligence and machine learning: These computational approaches will help predict modification patterns, identify regulatory networks, and generate testable hypotheses about HIST1H2BC function.

• Spatial transcriptomics and proteomics: These technologies will connect HIST1H2BC modifications to gene expression patterns with spatial resolution in tissues.

The integration of these technologies with antibody-based detection methods will significantly advance our understanding of HIST1H2BC's role in chromatin biology and disease mechanisms, potentially revealing new therapeutic strategies targeting histone-dependent processes.

How can researchers ensure reproducibility when working with HIST1H2BC (Ab-12) Antibody across different experimental systems?

Ensuring reproducibility when working with HIST1H2BC (Ab-12) Antibody requires systematic attention to several key aspects of experimental design and execution:

• Antibody validation in each experimental system:

  • Perform Western blot analysis in each cell line or tissue type

  • Include positive controls (cell lines known to express HIST1H2BC) and negative controls (HIST1H2BC-depleted cells)

  • Document lot-to-lot variation by testing different antibody batches

• Standardized protocols and reporting:

  • Document detailed protocols including antibody concentration, incubation times, and buffer compositions

  • Report catalog number, lot number, and supplier information in publications

  • Specify the exact epitope recognized by the antibody (peptide sequence around Lys12)

• Multiple detection methods:

  • Confirm findings using at least two independent techniques (e.g., Western blot and immunofluorescence)

  • When possible, use a second antibody targeting a different epitope

  • Validate key findings with orthogonal approaches (e.g., mass spectrometry)

• Biological and technical replicates:

  • Perform at least three independent biological replicates

  • Include multiple technical replicates within each experiment

  • Apply appropriate statistical analyses to assess variability and significance

• Comprehensive controls:

  • Include peptide competition controls to demonstrate specificity

  • Use recombinant HIST1H2BC as positive controls where applicable

  • Implement siRNA/shRNA knockdown controls to validate specificity

• Data sharing practices:

  • Deposit raw data in public repositories when possible

  • Share detailed protocols via protocol repositories (e.g., protocols.io)

  • Provide access to analysis scripts and software parameters

• Collaborative validation:

  • Establish multi-laboratory validation for critical findings

  • Participate in antibody validation consortia and databases

  • Compare results with published literature and address discrepancies