HIST1H2BC (Ab-12) Antibody

What is HIST1H2BC and what cellular processes is it involved in?

HIST1H2BC is a member of the histone H2B family, which plays a crucial role in packaging DNA into chromatin. This histone variant is essential for maintaining chromatin structure and stability, directly influencing gene expression regulation and DNA-based processes. The protein functions as a core component of nucleosomes, the fundamental repeating units of chromatin, and its post-translational modifications contribute to the histone code that regulates chromatin accessibility. HIST1H2BC's expression and modifications are implicated in various cellular processes including transcription, DNA replication, and DNA repair mechanisms. Dysregulation of HIST1H2BC has been associated with several diseases, including cancer and developmental disorders .

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

The HIST1H2BC (Ab-12) Antibody (PACO60470) has been validated for multiple research applications, including:

• Western Blotting (WB): Recommended dilution range of 1:100-1:1000

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

• Chromatin Immunoprecipitation (ChIP): For studying protein-DNA interactions

These applications enable researchers to detect, quantify, and analyze HIST1H2BC expression and interactions in various experimental contexts. The antibody has been specifically tested with positive Western blot detection in multiple cell lines (HeLa, HL60, MCF-7, NIH/3T3, K562) and tissues (rat liver, mouse kidney), making it versatile for cross-species studies in human, mouse, and rat samples .

What is the recommended protocol for Western blotting with HIST1H2BC (Ab-12) Antibody?

For optimal Western blotting results with the HIST1H2BC (Ab-12) Antibody, follow this protocol:

• Sample preparation: Prepare whole cell lysates from your cells of interest in an appropriate lysis buffer containing protease inhibitors.

• Protein separation: Load 20-50 μg of protein per lane and separate by SDS-PAGE.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

• Blocking: Block the membrane in 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute HIST1H2BC antibody to 1:100-1:1000 in blocking buffer and incubate overnight at 4°C.

• Washing: Wash the membrane 3-5 times with TBST, 5 minutes each.

• Secondary antibody: Incubate with anti-rabbit IgG secondary antibody (1:50000 dilution) for 1 hour at room temperature.

• Final washing: Wash 3-5 times with TBST, 5 minutes each.

• Detection: Apply ECL substrate and image the membrane.

The expected band size for HIST1H2BC is approximately 14 kDa. When troubleshooting, remember that histone proteins are relatively small and may require optimized gel conditions for proper separation .

How should the HIST1H2BC (Ab-12) Antibody be stored and handled?

For maximum stability and activity retention of the HIST1H2BC (Ab-12) Antibody:

• Storage temperature: Store at -20°C for long-term storage.

• Working aliquots: To avoid repeated freeze-thaw cycles, prepare small working aliquots before freezing.

• Storage buffer: The antibody is supplied in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability.

• Thawing procedure: Thaw on ice and mix gently by flicking the tube rather than vortexing.

• Short-term storage: For continuous use within 2-3 weeks, the antibody can be stored at 4°C.

• Transportation: Transport on ice packs or dry ice for longer shipments.

Proper storage and handling procedures are essential to maintain antibody specificity and sensitivity throughout your research applications .

How does HIST1H2BC relate to DNA replication processes?

Recent research has established significant connections between histone H2B variants, including HIST1H2BC, and DNA replication processes. H2B monoubiquitylation (H2Bub1) has been specifically implicated in regulating DNA replication in several ways:

• Replication origin association: H2Bub1 is present in chromatin adjacent to origins of DNA replication.

• Replication fork stability: H2Bub1 is maintained on daughter strands during replication by the Bre1 ubiquitin ligase.

• Fork progression: In the absence of H2Bub1, replication fork progression is significantly slowed.

• Replisome stability: H2Bub1 contributes to the stability of the replisome, particularly under replication stress conditions (e.g., hydroxyurea treatment).

• Nucleosome assembly: H2Bub1 promotes the assembly or stability of nucleosomes on newly replicated DNA.

These findings suggest that using HIST1H2BC antibodies to study H2B variants and their modifications can provide valuable insights into replication dynamics and genomic stability mechanisms. This is particularly relevant for research on cancer, where replication stress is a common feature .

What is the relationship between HIST1H2BC modifications and chromatin structure?

HIST1H2BC undergoes various post-translational modifications that significantly impact chromatin structure and function:

• Ubiquitylation: H2B ubiquitylation (particularly at lysine residues) influences transcription elongation, mRNA processing, and DNA replication. The absence of H2B ubiquitylation affects nucleosome assembly on newly replicated DNA.

• Acetylation: Acetylation of H2B at lysine residues (including Lys12) generally promotes a more open chromatin conformation, facilitating processes like transcription and DNA repair.

• Chromatin compaction: Modifications of HIST1H2BC directly affect the higher-order structure of chromatin, influencing accessibility to transcription factors and DNA repair machinery.

• Cross-talk with other histone modifications: HIST1H2BC modifications interact with modifications on other histones, creating a complex regulatory network. For example, H2B ubiquitylation influences H3K4 and H3K79 methylation.

Understanding these relationships is crucial for epigenetic research and can be studied using combination approaches with HIST1H2BC antibodies and other histone modification-specific antibodies .

What experimental approaches can be used to study HIST1H2BC's role in DNA damage response?

To investigate HIST1H2BC's involvement in DNA damage response, consider these methodological approaches:

• ChIP-sequencing with HIST1H2BC antibody:

  • Before and after DNA damage induction (e.g., UV, hydroxyurea, or chemical agents)

  • Map genome-wide distribution changes of HIST1H2BC

  • Compare with γ-H2AX distribution (DNA damage marker)

• Proximity ligation assays (PLA):

  • Detect interactions between HIST1H2BC and DNA repair proteins

  • Visualize spatial and temporal dynamics during damage response

• CRISPR/Cas9-mediated HIST1H2BC mutation:

  • Create cells with mutations at specific modification sites

  • Assess impact on recruitment of repair factors

  • Measure DNA repair kinetics using comet assay or repair factor foci resolution

• Immunofluorescence time-course studies:

  • Use HIST1H2BC antibody in combination with repair factor antibodies

  • Track co-localization at DNA damage sites over time

• Mass spectrometry analysis:

  • Identify damage-induced changes in HIST1H2BC post-translational modifications

  • Compare modification patterns in different damage response pathways

These approaches, used individually or in combination, can provide comprehensive insights into how HIST1H2BC contributes to maintaining genome integrity following DNA damage .

How can the HIST1H2BC (Ab-12) Antibody be used in multiplex immunofluorescence applications?

For advanced multiplex immunofluorescence applications, optimize your HIST1H2BC antibody protocol as follows:

• Sample preparation optimization:

  • For tissue sections: Use antigen retrieval methods specific for revealing nuclear epitopes (heat-induced epitope retrieval in citrate buffer, pH 6.0)

  • For cultured cells: Test different fixation methods (4% paraformaldehyde for 10-15 minutes often works well for nuclear proteins)

• Multiplex antibody panel design:

  • Primary antibody combination: Pair HIST1H2BC (Ab-12) rabbit antibody with mouse or goat antibodies against other targets

  • Ensure antibodies are raised in different host species to avoid cross-reactivity

  • Validate the specificity of each antibody individually before combining

• Sequential staining protocol:

  • Apply HIST1H2BC antibody at 1:100 dilution

  • Incubate overnight at 4°C in humidity chamber

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Include careful washing steps between antibody applications

  • Consider tyramide signal amplification for weak signals

• Controls and troubleshooting:

  • Include single-stained controls for each antibody

  • Use spectral unmixing if available

  • Block with appropriate sera between sequential stainings

  • Optimize order of antibody application (typically most robust signal last)

This approach enables simultaneous visualization of HIST1H2BC alongside other proteins of interest, facilitating studies of co-localization and functional relationships in complex cellular processes .

What are common issues when working with HIST1H2BC antibodies and how can they be resolved?

When working with HIST1H2BC antibodies, researchers commonly encounter several technical challenges:

These troubleshooting strategies are based on empirical evidence from multiple research laboratories working with histone antibodies and can significantly improve experimental outcomes .

How do I validate the specificity of the HIST1H2BC (Ab-12) Antibody for my experimental system?

To thoroughly validate the specificity of the HIST1H2BC (Ab-12) Antibody for your particular experimental system, implement this comprehensive validation strategy:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific signals should disappear in the blocked sample

• Genetic validation:

  • Use CRISPR/Cas9 to knock down HIST1H2BC

  • Compare antibody signals between wild-type and knockdown samples

  • Specific signals should be reduced in knockdown samples

• Orthogonal method comparison:

  • Compare results using alternative detection methods (e.g., mass spectrometry)

  • Use a different antibody targeting a different epitope of HIST1H2BC

  • Results should correlate across different detection methods

• Cross-species reactivity assessment:

  • Test the antibody on samples from different species with known sequence homology

  • Compare signal patterns with predicted conservation of the epitope

  • Signal intensity should correlate with epitope conservation

• Post-translational modification interference testing:

  • Treat samples to modify HIST1H2BC (e.g., phosphatase treatment)

  • Compare antibody binding before and after treatment

  • Determine if the antibody recognition is affected by modifications

This systematic validation approach ensures reliable and reproducible results across different experimental settings and biological systems .

How do HIST1H2BC antibodies perform in different cell types and species?

The performance of HIST1H2BC antibodies varies across cell types and species due to several factors:

• Species cross-reactivity:

  • The HIST1H2BC (Ab-12) Antibody shows reactivity with human, mouse, and rat samples

  • Signal strength generally correlates with sequence conservation at the epitope region

  • Human samples typically show the strongest signals due to exact epitope matching

• Cell type-specific considerations:

  • Expression levels: HIST1H2BC expression varies by cell type, with higher expression in rapidly dividing cells

  • Background signals: Different cell types may express varying levels of other H2B variants that could cross-react

  • Nuclear extraction efficiency: Varies between cell types based on nuclear membrane characteristics

• Validated positive controls:

  • HeLa, HL60, MCF-7, NIH/3T3, and K562 cell lines have shown positive Western blot results

  • Rat liver tissue and mouse kidney tissue have been validated for tissue applications

• Performance optimization by sample type:

  • Cell lines: Standard lysis protocols work well, with 1:500 dilution recommended

  • Primary cells: May require gentler lysis conditions and increased antibody concentration (1:200)

  • Tissue samples: Require thorough homogenization and potentially higher antibody concentration (1:100)

  • Embryonic samples: May show different histone variant patterns requiring careful interpretation

This information helps researchers select appropriate positive controls and optimize protocols based on their specific experimental model .

What are the best practices for quantifying HIST1H2BC levels in experimental samples?

For accurate quantification of HIST1H2BC levels across experimental conditions, adhere to these best practices:

• Sample preparation standardization:

  • Use consistent cell numbers or tissue amounts

  • Standardize lysis conditions and buffer compositions

  • Process all experimental samples simultaneously

• Western blot quantification:

  • Include a dilution series of a reference sample for standard curve generation

  • Use appropriate loading controls (total histone H4 or H3 are preferable to typical housekeeping proteins)

  • Ensure signal is within linear detection range of your imaging system

  • Analyze band intensities using software like ImageJ with background subtraction

  • Normalize HIST1H2BC signal to loading control

• ELISA-based quantification:

  • Use purified recombinant HIST1H2BC for standard curve

  • Apply samples at multiple dilutions to ensure values fall within standard curve

  • Plate samples in triplicate to assess technical variability

  • Calculate concentrations using four-parameter logistic regression

• Flow cytometry quantification:

  • Include calibration beads with known antibody binding capacity

  • Perform parallel staining of a reference sample across experiments

  • Use median fluorescence intensity for comparisons

  • Ensure proper compensation if performing multicolor analysis

• Statistical considerations:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Report both fold changes and absolute values when possible

  • Include measures of dispersion (standard deviation or standard error)

These methodologies enable reliable quantitative comparisons of HIST1H2BC levels across different experimental conditions, cell types, or treatment regimens .

How is HIST1H2BC implicated in cancer research and what methodologies are most appropriate?

HIST1H2BC and its modifications have emerging roles in cancer biology, with several key implications and recommended methodological approaches:

• Expression pattern alterations:

  • Multiple cancers show dysregulation of histone variant expression

  • Methodological approach: Use HIST1H2BC antibody for immunohistochemistry on tissue microarrays containing matched tumor and normal tissues

  • Analysis technique: Quantify nuclear staining intensity and correlation with clinical outcomes

• Post-translational modification changes:

  • Cancer cells frequently exhibit altered histone modification patterns

  • Methodological approach: ChIP-seq using HIST1H2BC antibody combined with modification-specific antibodies

  • Analysis technique: Identify cancer-specific changes in HIST1H2BC modification landscapes and associated genes

• DNA replication stress response:

  • Cancer cells experience heightened replication stress

  • HIST1H2BC modifications (particularly ubiquitylation) influence replication fork stability

  • Methodological approach: DNA fiber assay combined with HIST1H2BC antibody immunofluorescence

  • Analysis technique: Measure replication fork progression rates in cancer vs. normal cells

• Therapeutic targeting implications:

  • Enzymes modifying HIST1H2BC are potential therapeutic targets

  • Methodological approach: Combine HIST1H2BC antibody-based assays with small molecule inhibitors of histone-modifying enzymes

  • Analysis technique: Monitor changes in HIST1H2BC modification status and correlate with cellular responses

This research direction has significant potential for developing epigenetic biomarkers and therapeutic approaches targeting the chromatin landscape in cancer .

What role does HIST1H2BC play in epigenetic regulation and how can this be studied?

HIST1H2BC contributes significantly to epigenetic regulation through multiple mechanisms that can be studied using specialized techniques:

• Chromatin accessibility regulation:

  • HIST1H2BC modifications (especially acetylation at Lys12) alter chromatin compaction

  • Study method: Combine HIST1H2BC antibody ChIP with ATAC-seq or DNase-seq

  • Analysis approach: Correlate HIST1H2BC occupancy and modification status with chromatin accessibility maps

• Transcriptional regulation:

  • H2B ubiquitylation influences transcription elongation and mRNA processing

  • Study method: ChIP-seq with HIST1H2BC antibody followed by RNA-seq

  • Analysis approach: Integrate datasets to identify genes whose expression correlates with HIST1H2BC occupancy or modification patterns

• Inheritance of chromatin states:

  • During DNA replication, HIST1H2BC patterns must be maintained or reset

  • Study method: HIST1H2BC antibody ChIP at different cell cycle stages (synchronize cells)

  • Analysis approach: Track HIST1H2BC occupancy and modifications through cell division

• Cross-talk with other histone modifications:

  • H2B ubiquitylation influences H3K4 and H3K79 methylation

  • Study method: Sequential ChIP (first with HIST1H2BC antibody, then with antibodies against other modifications)

  • Analysis approach: Identify genomic regions with co-occurrence of multiple modifications

• Interaction with chromatin remodeling complexes:

  • HIST1H2BC interacts with various chromatin modifiers and remodelers

  • Study method: Co-immunoprecipitation with HIST1H2BC antibody followed by mass spectrometry

  • Analysis approach: Identify and validate protein complexes associated with HIST1H2BC in different cellular contexts

These approaches provide comprehensive insights into how HIST1H2BC contributes to the epigenetic landscape and cellular memory mechanisms .

How can HIST1H2BC antibodies be used to study the relationship between chromatin structure and DNA damage repair?

To investigate the relationship between HIST1H2BC, chromatin structure, and DNA damage repair, implement these specialized experimental approaches:

• Chromatin accessibility dynamics during repair:

  • Method: Combine HIST1H2BC ChIP-seq with ATAC-seq before and after damage induction

  • Analysis: Track changes in HIST1H2BC occupancy relative to chromatin opening at damage sites

  • Expected outcome: Identification of HIST1H2BC redistribution patterns during repair process

• Modification-specific recruitment of repair factors:

  • Method: Immunoprecipitate HIST1H2BC using the antibody, then perform Western blot for specific modifications and associated repair proteins

  • Analysis: Compare modification patterns and protein interactions before and after damage

  • Expected outcome: Identification of damage-induced HIST1H2BC modifications that recruit specific repair factors

• Real-time dynamics of HIST1H2BC at damage sites:

  • Method: Combine laser microirradiation with live-cell imaging of fluorescently tagged repair factors and subsequent HIST1H2BC immunofluorescence

  • Analysis: Track temporal recruitment and correlation between HIST1H2BC and repair factors

  • Expected outcome: Determination of whether HIST1H2BC precedes or follows repair factor recruitment

• Heterochromatin vs. euchromatin repair differences:

  • Method: ChIP with HIST1H2BC antibody followed by sequencing, with parallel staining for heterochromatin markers

  • Analysis: Compare HIST1H2BC dynamics in heterochromatic vs. euchromatic regions following damage

  • Expected outcome: Insight into chromatin-type specific repair mechanisms involving HIST1H2BC

• Cell cycle-specific relationships:

  • Method: Synchronize cells, induce damage at specific cell cycle stages, and perform HIST1H2BC antibody ChIP

  • Analysis: Compare HIST1H2BC patterns across cell cycle phases after damage

  • Expected outcome: Understanding of how cell cycle position influences HIST1H2BC's role in repair

These methodologies collectively provide a comprehensive framework for understanding how HIST1H2BC contributes to maintaining genome integrity through chromatin-based mechanisms .

What are emerging techniques that may enhance HIST1H2BC research?

Several cutting-edge technologies are poised to revolutionize HIST1H2BC research in the near future:

• CUT&Tag and CUT&RUN techniques:

  • These approaches offer higher signal-to-noise ratios than traditional ChIP

  • HIST1H2BC antibodies could be adapted for these protocols

  • Benefits include lower cell input requirements and improved spatial resolution

• Single-cell epigenomic profiling:

  • Emerging techniques allow histone modification analysis at single-cell resolution

  • HIST1H2BC antibodies could be incorporated into single-cell CUT&Tag workflows

  • This would reveal cell-to-cell variability in HIST1H2BC patterns within populations

• Super-resolution microscopy:

  • Techniques like STORM and PALM can visualize chromatin at nanometer resolution

  • Combined with HIST1H2BC antibody immunofluorescence, these approaches can reveal previously invisible spatial organization

  • Enables studies of HIST1H2BC distribution in chromatin domains

• Proximity labeling approaches:

  • Techniques like BioID or APEX2 fused to HIST1H2BC

  • Can identify proteins in proximity to HIST1H2BC in living cells

  • Provides temporal and spatial information about HIST1H2BC interactome

• Cryo-electron tomography:

  • Visualizes macromolecular complexes in their native cellular environment

  • Could be combined with immunogold labeling using HIST1H2BC antibodies

  • May reveal structural organization of HIST1H2BC-containing chromatin

These emerging technologies will enable researchers to address previously intractable questions about HIST1H2BC function and regulation in diverse biological contexts .

How do recent findings about HIST1H2BC inform therapeutic approaches in chromatin-related diseases?

Recent discoveries about HIST1H2BC biology highlight several promising therapeutic avenues for chromatin-related diseases:

• Targeting H2B ubiquitylation machinery:

  • Recent findings show H2B ubiquitylation influences DNA replication and repair

  • Inhibitors of E3 ligases (like Bre1) or deubiquitinases could modulate these processes

  • Potential applications in cancers with replication stress vulnerabilities

  • Methodological consideration: Use HIST1H2BC antibodies to monitor treatment effects on modification status

• Exploiting synthetic lethality:

  • Cells lacking proper H2B ubiquitylation show impaired replication fork stability

  • These cells may be hypersensitive to certain DNA-damaging agents

  • Potential for combination therapies targeting both H2B modification and DNA repair

  • Methodological consideration: Use HIST1H2BC antibodies in patient-derived xenograft models to stratify potential responders

• Acetylation modulation approaches:

  • H2B acetylation (including at Lys12) regulates chromatin accessibility

  • HDAC inhibitors and HAT activators could be used to target this modification

  • Potentially useful in transcriptional reprogramming strategies

  • Methodological consideration: Monitor acetylation status with specific antibodies alongside HIST1H2BC occupancy

• Diagnostic and prognostic applications:

  • HIST1H2BC modifications as biomarkers for disease stratification

  • ChIP-seq using HIST1H2BC antibodies could identify patient-specific epigenetic signatures

  • Potential for precision medicine approaches based on chromatin patterns

  • Methodological consideration: Develop standardized clinical assays using HIST1H2BC antibodies

These therapeutic strategies represent translational applications of fundamental research on HIST1H2BC biology and highlight the importance of continuing to develop specific antibodies and assays for histone variants and their modifications .