HIST1H2BC (Ab-5) Antibody

What is HIST1H2BC and what role does it play in cellular function?

HIST1H2BC is a core histone protein belonging to the H2B family, which is crucial for packaging DNA into chromatin. It plays a significant role in gene regulation and chromatin structure, making it a key player in epigenetic processes. This histone variant is involved in the organization of nucleosomes and contributes to the regulation of DNA accessibility during transcription. Dysregulation of histone proteins like HIST1H2BC has been implicated in diseases such as cancer, where aberrant gene expression contributes to tumorigenesis . Understanding HIST1H2BC function is essential for unraveling the molecular mechanisms underlying various cellular processes and disease development.

What are the key specifications of the HIST1H2BC (Ab-5) Antibody?

The HIST1H2BC (Ab-5) Antibody (PACO65127) is a highly specific rabbit polyclonal antibody targeting human HIST1H2BC. The antibody has been developed using a synthesized peptide derived from Human Histone H2B type 1-C/E/F/G/I protein, specifically amino acids 2-13. It is supplied as a 50μL liquid preparation in a buffer containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative. The antibody has been purified using antigen affinity methods and validated for ELISA and IHC applications, with recommended dilutions of 1:10-1:100 for IHC applications .

How does HIST1H2BC differ from other histone variants, and why is this important for experimental design?

HIST1H2BC is part of the histone H2B family but has specific sequence variations that distinguish it from other H2B variants. These differences may influence nucleosome stability, chromatin packaging, and interactions with chromatin remodeling complexes. When designing experiments, researchers must consider these variant-specific properties as they might affect:

• DNA binding affinity and nucleosome stability

• Post-translational modification patterns

• Interactions with non-histone proteins

• Tissue-specific expression patterns

• Role in specific cellular processes

For accurate results, experiments should use antibodies with high specificity for the HIST1H2BC variant, such as the Ab-5 antibody, which targets a unique epitope (amino acids 2-13) of the protein . Cross-reactivity with other histone variants could lead to misinterpretation of results, especially in complex tissues or experimental conditions.

What are the validated applications for HIST1H2BC (Ab-5) Antibody, and what optimization steps are recommended?

The HIST1H2BC (Ab-5) Antibody has been validated for ELISA and immunohistochemistry (IHC) applications . For optimal results in these applications, researchers should consider the following optimization steps:

For IHC applications:

• Begin with the recommended dilution range (1:10-1:100) and optimize for your specific tissue

• Include appropriate positive and negative controls

• Optimize antigen retrieval methods (heat-induced or enzymatic)

• Test different blocking solutions to minimize background

• Adjust incubation times and temperatures

For ELISA applications:

• Start with higher dilutions (1:1000-1:5000) and adjust based on signal strength

• Optimize coating concentration of capture reagents

• Test different blocking buffers to improve signal-to-noise ratio

• Adjust washing protocols to reduce background

While not explicitly validated, researchers might also consider testing this antibody for Western blot, ChIP, or immunofluorescence applications with proper controls and optimization.

How should researchers prepare samples for optimal detection of HIST1H2BC using the Ab-5 antibody?

Sample preparation is critical for successful detection of HIST1H2BC. For various applications, consider these methodological guidelines:

For tissue samples (IHC):

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process tissues through standard paraffin embedding

• Section tissues at 4-6 μm thickness

• For antigen retrieval, use citrate buffer (pH 6.0) with heat treatment

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Use protein blocking solutions to minimize non-specific binding

For cell lysates (potential Western blot application):

• Harvest cells at 70-80% confluence

• Lyse cells in a buffer containing protease inhibitors and DNase

• Include 0.1% SDS in lysis buffer to aid histone extraction

• Sonicate samples to shear DNA and release chromatin-bound proteins

• Determine protein concentration and load equal amounts

• Include acidic extraction methods for enriching histone proteins

For both preparations, it's essential to include controls to validate antibody specificity and performance in your experimental system .

What controls should be included when using HIST1H2BC (Ab-5) Antibody in research experiments?

Proper controls are essential for validating results with the HIST1H2BC (Ab-5) Antibody:

• Positive Control: Use cell lines or tissues known to express HIST1H2BC (such as HeLa or 293 cells)

• Negative Control:

  • Tissue or cells known not to express HIST1H2BC

  • Isotype control (rabbit IgG at the same concentration)

  • Secondary antibody-only control to assess non-specific binding

• Peptide Competition/Blocking Control:

  • Pre-incubate the antibody with excess HIST1H2BC peptide (2-13aa)

  • This should abolish specific staining if the antibody is specific

• siRNA/shRNA Knockdown Control:

  • Compare staining in cells with and without HIST1H2BC knockdown

  • Should show reduced signal in knockdown samples

• Recombinant Protein Control:

  • Test antibody against recombinant HIST1H2BC protein

  • Useful for confirming specificity and determining sensitivity

These controls help validate antibody specificity and ensure that observed signals are truly representative of HIST1H2BC expression or localization .

What are common issues encountered when using HIST1H2BC (Ab-5) Antibody, and how can they be resolved?

Researchers may face several challenges when working with HIST1H2BC (Ab-5) Antibody:

• Weak or No Signal:

  • Increase antibody concentration (try the higher end of recommended dilution)

  • Optimize antigen retrieval (extend time or try different pH buffers)

  • Ensure fresh samples and proper storage of the antibody

  • Check for protein degradation in samples

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

• High Background:

  • Increase blocking time and use more stringent blocking agents

  • Use more dilute antibody concentration

  • Ensure thorough washing steps

  • Check for endogenous peroxidase or phosphatase activity

  • Use more specific detection systems

• Non-specific Binding:

  • Increase antibody dilution

  • Optimize blocking conditions

  • Validate with peptide competition assays

  • Pre-absorb antibody with non-specific proteins

• Inconsistent Results:

  • Standardize sample preparation and experimental protocols

  • Ensure antibody storage conditions are maintained

  • Use the same lot number for comparative experiments

  • Control for cell confluence and growth conditions

For all troubleshooting, comparing your protocols with those in published literature using this or similar antibodies can provide valuable insights .

How can researchers optimize detection sensitivity for low-abundance HIST1H2BC?

Detecting low-abundance HIST1H2BC requires optimization strategies:

• Signal Amplification Methods:

  • Use tyramide signal amplification (TSA) systems

  • Employ biotin-streptavidin amplification

  • Utilize polymer-based detection systems

• Sample Preparation Optimization:

  • Enrich for nuclear fractions in cell lysates

  • Use histone extraction protocols with acidic conditions

  • Optimize chromatin immunoprecipitation (ChIP) protocols

• Instrument Settings:

  • Increase exposure time for imaging

  • Optimize gain and offset in fluorescence microscopy

  • Use more sensitive detection instruments

• Protocol Modifications:

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

  • Reduce washing stringency slightly (shorter washes)

  • Use higher antibody concentration within the recommended range

  • Consider using fresh samples as freeze-thaw cycles may decrease antigen detection

• Combined Approaches:

  • Use dual detection methods

  • Consider proximity ligation assays for detecting protein interactions

Each optimization approach should be tested systematically with appropriate controls to ensure specificity is maintained while sensitivity is enhanced .

How can HIST1H2BC (Ab-5) Antibody be utilized to study histone modifications and their role in gene regulation?

The HIST1H2BC (Ab-5) Antibody can be employed in sophisticated experimental designs to investigate histone modifications:

• Sequential ChIP (ChIP-reChIP):

  • First immunoprecipitate with HIST1H2BC antibody

  • Follow with a second immunoprecipitation using antibodies against specific modifications (e.g., acetylation, methylation)

  • This approach identifies genomic regions where HIST1H2BC carries specific modifications

• Mass Spectrometry Analysis:

  • Immunoprecipitate HIST1H2BC using the Ab-5 antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Identify post-translational modifications and interacting proteins

• ChIP-Seq Approaches:

  • Use HIST1H2BC antibody for ChIP followed by next-generation sequencing

  • Map genomic distribution of HIST1H2BC

  • Compare with datasets for histone modifications or transcription factors

• Correlation with Histone-Modifying Enzymes:

  • Study co-localization of HIST1H2BC with histone writers, readers, and erasers

  • Investigate changes in HIST1H2BC distribution after inhibiting specific enzymes

Research has shown connections between histone H2B ubiquitination and H3K4 methylation in epigenetic regulation. For instance, H2B ubiquitination recruits H3K4me3 through a process dependent on the 19S proteasome subunit RPT6, affecting gene transcription and memory formation . These approaches can reveal how HIST1H2BC participates in similar regulatory networks.

How does HIST1H2BC compare with other histone variants like HIST1H2BK in disease contexts?

Comparing HIST1H2BC with other histone variants reveals important differences in disease associations and functional roles:

• Disease Associations:

  • HIST1H2BC has been implicated in cancer development through its role in chromatin regulation and gene expression

  • HIST1H2BK has been specifically identified as a biomarker for predicting neoadjuvant-chemotherapy (NACT) response and prognosis in gastric cancer

• Functional Mechanisms:

  • While HIST1H2BC's specific role in disease pathogenesis is less defined in the provided search results, HIST1H2BK has been shown to inhibit 5-FU-induced apoptosis by upregulating A2M transcription and activating the LRP1/PI3K/Akt signaling pathway in gastric cancer cells

  • HIST1H2BK overexpression promotes 5-FU resistance and increased cell proliferation in gastric cancer cells

• Biomarker Potential:

  • HIST1H2BK has been validated as a plasma biomarker for predicting treatment response and prognosis

  • Similar studies on HIST1H2BC might reveal its potential as a biomarker in other cancer types or diseases

• Intercellular Communication:

  • HIST1H2BK-overexpressing 5-FU-resistant gastric cancer cells can transmit drug resistance to sensitive cells through secreted HIST1H2BK

  • Similar mechanisms might exist for HIST1H2BC but require further investigation

These comparisons highlight the importance of studying specific histone variants individually despite their structural similarities. Researchers should consider designing comparative studies to elucidate the distinct roles of HIST1H2BC versus other H2B variants in disease contexts.

How does HIST1H2BC (Ab-5) Antibody compare with HIST1H2BC (Ab-108) Antibody for specific applications?

Understanding the differences between HIST1H2BC antibodies targeting different epitopes is crucial for selecting the most appropriate reagent:

Application-specific considerations:

• For Western Blotting: Ab-108 has been specifically validated for WB applications with positive detection in multiple cell lines (HeLa, 293, HepG2, HL60, MCF-7)

• For Immunoprecipitation: Only Ab-108 is validated for IP applications, making it the preferred choice for protein interaction studies

• For Structural Studies: Ab-5 targets the N-terminal region (aa 2-13), which may be more accessible in certain chromatin states, while Ab-108 targets a region around Lysine 108, which might be involved in specific post-translational modifications

• For Detecting Modified Forms: The epitope locations differ significantly, which may affect detection of modified HIST1H2BC. If studying modifications near Lys-108, Ab-5 would be preferable to avoid epitope masking issues

Choose between these antibodies based on your specific application and the region of HIST1H2BC that is most relevant to your research question .

What are the key considerations when selecting between different histone H2B antibodies for epigenetic research?

When selecting antibodies for histone H2B research, consider these critical factors:

• Variant Specificity:

  • Determine whether you need an antibody specific to HIST1H2BC or one that recognizes multiple H2B variants

  • Examine the immunogen sequence and test for cross-reactivity with other H2B variants

  • Consider the biological context and whether variant specificity is critical to your research question

• Epitope Location:

  • N-terminal antibodies (like Ab-5) may be better for detecting histones in intact chromatin

  • C-terminal or internal epitope antibodies may be better for detecting specific modifications

  • If studying a specific post-translational modification, ensure the antibody's epitope doesn't overlap with that site

• Validated Applications:

  • Match the antibody to your intended application (ChIP, WB, IHC, IF, ELISA)

  • Review validation data for similar experimental conditions to yours

  • Consider testing multiple antibodies targeting different epitopes for critical experiments

• Modification State:

  • Some antibodies may preferentially recognize modified or unmodified forms

  • Check whether the antibody can detect ubiquitinated, acetylated, or methylated histone forms

  • For modification-specific detection, use antibodies raised against the modified peptide

• Chromatin Context:

  • Consider whether your experiment involves condensed chromatin, open chromatin, or extracted histones

  • Epitope accessibility varies depending on chromatin state and experimental conditions

A comprehensive approach might include using multiple antibodies targeting different epitopes or modifications to build a complete picture of HIST1H2BC dynamics in your experimental system .

How should researchers interpret changes in HIST1H2BC levels in the context of epigenetic regulation?

Interpreting changes in HIST1H2BC levels requires consideration of multiple factors:

• Context-Dependent Interpretation:

  • Increased HIST1H2BC might indicate chromatin remodeling, cell cycle changes, or compensatory mechanisms

  • Decreased levels could suggest transcriptional repression, histone replacement, or targeted degradation

  • Always interpret changes in relation to cellular context (e.g., differentiation, stress response, disease state)

• Correlation with Other Epigenetic Marks:

  • Changes in HIST1H2BC should be interpreted alongside changes in histone modifications (e.g., H3K4me3, H3K27me3)

  • H2B ubiquitination has been shown to control H3K4me3 levels through histone cross-talk mechanisms

  • Use sequential ChIP or co-staining approaches to determine relationships between HIST1H2BC and other marks

• Genomic Distribution:

  • Global versus gene-specific changes have different implications

  • ChIP-seq analysis can reveal whether changes occur at specific genomic regions

  • Correlate with transcriptional activity data to understand functional consequences

• Temporal Dynamics:

  • Transient versus sustained changes suggest different regulatory mechanisms

  • For example, learning-induced increases in H2B ubiquitination return to baseline after 24 hours, suggesting a role in the memory consolidation process

• Physiological Relevance:

  • Similar to how H2B ubiquitination increases in area CA1 of the hippocampus after contextual fear conditioning , HIST1H2BC changes may have tissue-specific and stimulus-specific patterns

  • These patterns might reflect underlying physiological processes

Integrate multiple lines of evidence to build a comprehensive interpretation of HIST1H2BC dynamics in your experimental system.

What statistical approaches are recommended for analyzing HIST1H2BC expression or modification data?

Rigorous statistical analysis is essential for interpreting HIST1H2BC data:

• For Expression Level Analysis:

  • Use ANOVA followed by appropriate post-hoc tests for multiple group comparisons

  • Apply t-tests (paired or unpaired) for two-group comparisons

  • Consider non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) if data is not normally distributed

  • Normalize to appropriate housekeeping proteins or total histone H2B levels

• For ChIP and ChIP-seq Analysis:

  • Use enrichment over input or IgG control for ChIP-qPCR

  • For ChIP-seq, apply specialized packages like MACS2 for peak calling

  • Consider differential binding analysis using DESeq2 or edgeR

  • Perform gene ontology and pathway enrichment analysis for identified target genes

• For Correlation Analysis:

  • Use Pearson's or Spearman's correlation to associate HIST1H2BC levels with other markers

  • Apply multivariate analyses to control for confounding factors

  • Consider integrative approaches that combine ChIP-seq, RNA-seq, and proteomic data

• For Image Analysis:

  • Use quantitative image analysis software for immunofluorescence or IHC

  • Apply appropriate background subtraction methods

  • Consider cell-by-cell analysis rather than whole-field measurements

  • Use coefficient of variation to assess staining heterogeneity

• Sample Size and Power:

  • Conduct power analysis to determine appropriate sample sizes

  • Report effect sizes alongside p-values

  • Control for multiple testing using Benjamini-Hochberg or similar approaches

These statistical approaches should be tailored to your specific experimental design and data type, ensuring robust and reproducible findings.

How can researchers identify and validate HIST1H2BC-associated protein complexes and modifications?

Identifying and validating HIST1H2BC-associated complexes involves multiple complementary approaches:

• Immunoprecipitation and Mass Spectrometry:

  • Use HIST1H2BC (Ab-5) or (Ab-108) antibodies for immunoprecipitation

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate key interactions by reciprocal IP or proximity ligation assay

  • Identify post-translational modifications on immunoprecipitated HIST1H2BC

• ChIP-seq and Protein Complex Analysis:

  • Perform sequential ChIP (ChIP-reChIP) to identify co-occupancy with other factors

  • Compare HIST1H2BC ChIP-seq data with datasets for potential interacting partners

  • Use publicly available ChIP-seq datasets to identify transcription factors or chromatin modifiers that co-localize with HIST1H2BC

• Functional Validation:

  • Use CRISPR-Cas9 to knock out or modify HIST1H2BC or its interacting partners

  • Assess changes in chromatin structure, gene expression, or cellular phenotypes

  • Similar to studies on H2B ubiquitination, examine how HIST1H2BC modifications affect other histone marks

• Modification-Specific Analysis:

  • Use antibodies specific to modified HIST1H2BC (if available)

  • Apply mass spectrometry to identify and quantify post-translational modifications

  • Examine how enzymes like E3 ubiquitin ligases affect HIST1H2BC, similar to how the 19S proteasome subunit RPT6 influences H2B ubiquitination

• Bioinformatic Approaches:

  • Use motif analysis to predict protein-binding sites on HIST1H2BC

  • Apply machine learning algorithms to predict functional interactions

  • Integrate multi-omics data to build HIST1H2BC-centered regulatory networks

By combining these approaches, researchers can build a comprehensive understanding of HIST1H2BC-associated complexes and their functional significance in various cellular contexts.

What are emerging research areas involving HIST1H2BC that researchers should consider?

Several promising research directions for HIST1H2BC are emerging based on recent findings:

• Role in Neurodegenerative Diseases:

  • Given the importance of H2B ubiquitination in memory formation , investigate HIST1H2BC in neurodegenerative conditions

  • Explore how HIST1H2BC modifications change during aging and in conditions like Alzheimer's disease

  • Study how HIST1H2BC contributes to neuronal plasticity and memory consolidation

• Cancer Biomarker Development:

  • Similar to HIST1H2BK's role as a biomarker for chemotherapy response in gastric cancer , investigate HIST1H2BC as a potential biomarker

  • Examine HIST1H2BC levels in liquid biopsies from cancer patients

  • Study correlations between HIST1H2BC expression and treatment resistance

• Extracellular Histone Functions:

  • Investigate whether HIST1H2BC, like HIST1H2BK , can be secreted and function in cell-to-cell communication

  • Explore potential roles in immune response and inflammation

  • Study HIST1H2BC in extracellular vesicles and their functional implications

• Targeted Epigenetic Therapy:

  • Develop approaches to modulate HIST1H2BC modifications for therapeutic purposes

  • Explore how existing epigenetic drugs affect HIST1H2BC dynamics

  • Design PROTAC or similar approaches targeting HIST1H2BC-containing complexes

• Single-Cell Epigenomics:

  • Apply single-cell technologies to study HIST1H2BC heterogeneity in tissues

  • Integrate with spatial transcriptomics to understand tissue-specific roles

  • Examine cell-type-specific functions of HIST1H2BC in development and disease

These emerging areas represent significant opportunities for researchers to expand our understanding of HIST1H2BC biology and its implications for human health and disease.