HIST1H2BC (Ab-23) Antibody

What is HIST1H2BC and what are its biological functions?

HIST1H2BC encodes Histone H2B type 1-C/E/F/G/I, a core component of nucleosomes that plays a critical role in chromatin organization. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template. This structural organization means that histones like H2B play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The function of HIST1H2BC is regulated through post-translational modifications that form part of the "histone code" and through nucleosome remodeling processes that dynamically alter chromatin structure . Recent research has also implicated histone variants in conditions such as endocrine-resistant breast cancer, highlighting their potential significance in disease mechanisms .

How does the HIST1H2BC (Ab-23) Antibody differ from other histone H2B antibodies?

The HIST1H2BC (Ab-23) Antibody is distinguished by its specific targeting of the lysine 23 region of Histone H2B type 1-C/E/F/G/I. This specificity contrasts with other antibodies such as the HIST1H2BC (Ab-20) Antibody, which targets the lysine 20 region . Understanding these epitope differences is crucial as they may affect recognition of different post-translational modifications and functional states of the histone. The Ab-23 antibody is a rabbit-derived polyclonal IgG antibody with validated reactivity against human samples, primarily in ELISA and IHC applications . By comparison, other H2B antibodies like those referenced in the search results may have broader reactivity (human/mouse/rat) or different application profiles (Western blot, ChIP, Flow Cytometry) .

What experimental evidence validates the specificity of HIST1H2BC (Ab-23) Antibody?

The specificity of HIST1H2BC (Ab-23) Antibody derives from its development against a carefully selected immunogen: a peptide sequence surrounding lysine 23 of Human Histone H2B type 1-C/E/F/G/I . While the search results don't provide explicit cross-reactivity testing data for this specific antibody, the established methodology for validating histone antibodies typically includes Western blot analysis against purified histone proteins, as demonstrated for similar antibodies . Researchers working with this antibody should consider performing their own validation tests, including peptide competition assays and knockout/knockdown controls, to confirm specificity in their experimental systems.

What are the validated applications for HIST1H2BC (Ab-23) Antibody?

The HIST1H2BC (Ab-23) Antibody has been validated for two primary applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): With recommended dilutions of 1:2000 to 1:10000 .

• Immunohistochemistry (IHC): With recommended dilutions of 1:10 to 1:100 .

This application profile suggests the antibody is particularly valuable for quantitative protein detection and localization studies in tissue samples. By comparison, other histone H2B antibodies mentioned in the search results have been validated for additional applications including Western blot, immunoprecipitation, chromatin immunoprecipitation, flow cytometry, and immunofluorescence , indicating potential areas for expanded use of the Ab-23 antibody pending further validation.

What protocol optimizations improve signal detection in IHC applications?

For optimal IHC results with HIST1H2BC (Ab-23) Antibody, researchers should consider the following protocol optimizations:

• Antigen retrieval: Heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) is often effective for histone epitopes, as demonstrated in protocols for similar antibodies .

• Antibody concentration: Begin with the recommended dilution range (1:10 to 1:100) and optimize for your specific tissue type .

• Incubation conditions: Overnight incubation at 4°C may improve specific binding while reducing background.

• Detection system: Use a sensitive detection system appropriate for rabbit IgG antibodies.

• Blocking: Thorough blocking (e.g., with 5-10% normal serum from the same species as the secondary antibody) is critical to reduce background.

Similar histone antibodies have shown successful IHC staining using the Bond™ system with standardized protocols, suggesting this system could be effective for HIST1H2BC (Ab-23) as well .

What controls should be included when using HIST1H2BC (Ab-23) Antibody?

To ensure experimental rigor when using the HIST1H2BC (Ab-23) Antibody, the following controls should be included:

• Primary antibody omission control: To assess non-specific binding of the secondary detection system.

• Isotype control: Using a rabbit IgG isotype control antibody at the same concentration to identify any non-specific binding.

• Peptide competition control: Pre-incubating the antibody with the immunizing peptide (Lys-23 region) should abolish specific staining.

• Positive tissue control: Using tissues known to express HIST1H2BC at detectable levels.

• Negative tissue control: Using tissues with minimal HIST1H2BC expression.

• HIST1H2BC knockdown/knockout control: If available, this provides the most stringent specificity control.

For Western blot applications (if extended to this use), purified recombinant histone proteins can serve as controls, similar to the approach used for other H2B antibodies where specificity across histone family members was demonstrated .

What are common causes of background staining when using histone antibodies?

Background staining is a common challenge with histone antibodies due to the abundant nature and high conservation of these proteins. Common causes include:

• Insufficient blocking: Histones are basic proteins that can bind non-specifically to acidic cellular components.

• Overfixation: Excessive fixation can create artifactual epitopes or cause protein cross-linking that increases non-specific binding.

• Suboptimal antibody concentration: Using too high a concentration (lower dilution) of primary antibody often increases background.

• Cross-reactivity: Histone family members share significant sequence homology, potentially leading to recognition of multiple histone variants.

• Post-translational modifications: Modified histones may show differential recognition by the antibody.

To mitigate these issues, researchers should optimize blocking conditions (using BSA or serum), carefully control fixation times, titrate antibody concentrations, and consider using histone-specific blocking reagents.

How can specificity of HIST1H2BC (Ab-23) Antibody be validated?

To validate the specificity of HIST1H2BC (Ab-23) Antibody for your experimental system:

• Western blot against purified histones: Test against recombinant histones (H1, H2A, H2B, H3, H4) to confirm specific binding to H2B only, similar to validation done for other H2B antibodies .

• Peptide competition assays: Pre-incubation with the immunizing peptide should abolish specific signal.

• RNAi experiments: Signal should be reduced in cells with HIST1H2BC knockdown.

• Cross-reactivity assessment: Test reactivity against related histone variants.

• Mass spectrometry verification: For immunoprecipitation applications, MS analysis can confirm the identity of precipitated proteins.

These validation approaches will provide confidence in the specificity of results obtained with this antibody.

What storage conditions maintain antibody performance?

For optimal performance of HIST1H2BC (Ab-23) Antibody, follow these storage recommendations:

• Short-term storage: The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

• Temperature: Store at -20°C for long-term stability.

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles.

• Working dilutions: Prepare working dilutions immediately before use.

• Stability: Monitor stability with positive controls when using antibody from older lots.

These practices will help maintain antibody integrity and experimental reproducibility over time.

How can HIST1H2BC (Ab-23) Antibody contribute to epigenetic research?

The HIST1H2BC (Ab-23) Antibody, targeting a specific lysine residue (K23), offers potential value for epigenetic research in several ways:

• Monitoring post-translational modifications: Though the Ab-23 antibody itself recognizes the K23 region regardless of modification status, it could be used in combination with modification-specific antibodies to understand the interplay between different histone marks.

• Chromatin organization studies: Given that histones are fundamental to chromatin structure, this antibody could help investigate how HIST1H2BC contributes to higher-order chromatin organization in different cellular contexts.

• Gene regulation mechanisms: By examining the distribution of HIST1H2BC in relation to gene regulatory elements, researchers could gain insights into how this histone variant influences transcription.

• Disease mechanisms: As suggested by related research on histone variants in cancer , the antibody could help investigate the role of HIST1H2BC in disease processes.

For optimal results in chromatin immunoprecipitation studies, researchers might consider protocols similar to those established for other histone antibodies, which typically use 2.5 μl of antibody with 10 μg of chromatin (approximately 4 x 10^6 cells) per IP .

What is known about HIST1H2BC expression patterns in cancer research?

While the search results don't provide specific information about HIST1H2BC expression in cancer, they do mention related research on histone H2B variants:

• Endocrine-resistant breast cancer: Research indicates that the histone variant HIST1H2BE (closely related to HIST1H2BC) shows hypomethylation and increased expression in estrogen deprivation-resistant breast cancer cell lines compared to parental cells .

• Expression pattern complexity: Gene expression analysis revealed varied expression of HIST1H2BE across breast cancer cell lines, without restriction to specific molecular subtypes .

• Functional significance: Both overexpression and downregulation of HIST1H2BE caused decreased proliferation in breast cancer cell lines, suggesting the need for tightly controlled expression of this histone variant .

These findings on HIST1H2BE suggest that HIST1H2BC might also have complex roles in cancer biology, potentially warranting investigation with tools like the Ab-23 antibody. Researchers could use this antibody to examine HIST1H2BC expression patterns across tumor types and correlate with clinical outcomes.

How does ubiquitylation of H2B influence its function?

Ubiquitylation represents an important post-translational modification of histone H2B that influences chromatin dynamics and gene expression. According to the search results:

• Site-specificity: Ubiquitylation occurs at Lys120 of histone H2B .

• Detection specificity: Specialized antibodies like the Ubiquityl-Histone H2B (Lys120) antibody detect H2B only when ubiquitylated at this specific site .

• Functional significance: While not explicitly stated in the search results, histone H2B ubiquitylation is known to play roles in transcriptional elongation, DNA damage response, and cell cycle progression.

Researchers interested in studying the interplay between different histone modifications could investigate whether the region recognized by the HIST1H2BC (Ab-23) Antibody (around Lys23) shows any functional relationship with ubiquitylation at Lys120, potentially revealing new insights into the histone code.

How do antibody-based methods compare with genomic approaches for studying HIST1H2BC?

Researchers have multiple options for studying HIST1H2BC, each with distinct advantages:

Integrating multiple approaches provides the most comprehensive understanding of HIST1H2BC biology and function.

What alternative antibodies can be used to study H2B histones?

Researchers have several antibody options for H2B histone studies, each with specific characteristics:

This diversity of antibodies allows researchers to select the most appropriate tool based on their specific experimental questions, techniques, and sample types.

How might HIST1H2BC studies contribute to understanding cancer mechanisms?

Research on histone variants like HIST1H2BC has significant potential for advancing cancer research:

• Epigenetic dysregulation: The finding that histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant breast cancer suggests epigenetic mechanisms may regulate histone variant expression in cancer .

• Therapeutic resistance: HIST1H2BE's altered expression in aromatase inhibitor-resistant breast cancer cells points to potential roles for histone variants in therapeutic resistance mechanisms .

• Expression regulation: The observation that both overexpression and downregulation of HIST1H2BE decreased proliferation suggests a need for precisely controlled expression of histone variants in cancer cells .

• Biomarker potential: Analysis of HIST1H2BE mRNA expression in ER+ AI-treated breast tumors suggests possible prognostic or predictive value .

These findings provide a framework for similar investigations of HIST1H2BC in cancer biology, potentially using the Ab-23 antibody to examine protein expression patterns across cancer types and stages.

What emerging technologies could enhance HIST1H2BC research?

Several cutting-edge approaches could advance HIST1H2BC research:

• Single-cell technologies: Applying single-cell proteomics and ChIP-seq could reveal cell-to-cell variation in HIST1H2BC distribution and function, overcoming limitations of population-averaged data.

• CRISPR/Cas9 genome editing: Precise modification of HIST1H2BC could help dissect its specific roles in chromatin organization and gene regulation.

• Super-resolution microscopy: Techniques like STORM or PALM could visualize the spatial organization of HIST1H2BC within chromatin at nanometer resolution.

• Mass spectrometry-based proteomics: Advanced MS approaches could comprehensively map post-translational modifications on HIST1H2BC and identify interacting protein partners.

• Integrative multi-omics: Combining ChIP-seq, RNA-seq, and proteomics data could provide a holistic view of HIST1H2BC function in different cellular contexts.

These approaches, combined with antibody-based detection methods like those employing the HIST1H2BC (Ab-23) Antibody, will drive future discoveries about the role of this histone variant in normal biology and disease.