Histone H2B type 1-K Recombinant Monoclonal Antibody

What is Histone H2B type 1-K and what role does it play in chromatin biology?

Histone H2B type 1-K (HIST1H2BK) is a core component of the nucleosome, the fundamental unit of chromatin packaging in eukaryotic cells. As part of the histone octamer, H2B type 1-K helps wrap and compact DNA into chromatin, which regulates DNA accessibility to cellular machinery. This histone variant plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . Recent research has revealed that H2B type 1-K is not merely a structural component but also participates in specific biological processes, particularly in senescent cells, where it accumulates under conditions of persistent DNA damage .

How does Histone H2B type 1-K differ from other H2B variants?

Histone H2B type 1-K is one of several H2B variants that have been identified in mammalian genomes. While core H2B histones share high sequence homology, specific variants like H2B type 1-K have unique sequence features that enable distinct biological functions. Phylogenomic analyses have identified five major H2B variants broadly present in mammalian genomes: H2B.1, H2B.L (also called subH2B), H2B.W, and the more recently described H2B.K and H2B.N .

H2B type 1-K (HIST1H2BK) has a molecular weight of approximately 14 kDa and contains variant-specific amino acid sequences, particularly in its C-terminal region, which can be used for unambiguous identification via mass spectrometry . These subtle differences in amino acid composition contribute to specific chromatin structures and functions, particularly in cellular senescence pathways.

Why is Histone H2B type 1-K relevant for senescence and aging research?

Recent studies have revealed that H2B type 1-K accumulates specifically in deeply senescent cells that harbor persistent DNA damage. Using combined protein profiling and bottom-up mass spectrometry approaches, researchers demonstrated a two-fold increase in H2B type 1-K abundance in deep senescent conditions with persistent DNA damage, reaching almost 40% of total H2B content compared to control conditions .

This specific enrichment was not observed in quiescent cells or in cells induced into senescence without DNA damage, suggesting that H2B type 1-K may serve as a novel biomarker for senescent cells containing persistent DNA damage . This finding has significant implications for aging research, as cellular senescence is a key contributor to tissue aging and age-related pathologies. Researchers studying aging mechanisms may use H2B type 1-K antibodies to identify and quantify senescent cell populations in tissue samples.

How can Histone H2B type 1-K antibodies be used to study chromatin dynamics?

Histone H2B type 1-K antibodies provide a powerful tool to investigate chromatin composition and dynamics during various cellular processes. The specific recognition of H2B type 1-K allows researchers to track changes in histone variant composition under different physiological and pathological conditions.

For advanced chromatin studies, H2B type 1-K antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to identify genomic regions associated with this histone variant. The data can be analyzed using techniques such as ChIP-seq to generate genome-wide maps of H2B type 1-K distribution, providing insights into its role in transcriptional regulation and DNA damage responses .

When studying chromatin dynamics, researchers should consider comparing H2B type 1-K distribution with other histone variants and their post-translational modifications to develop a comprehensive understanding of the histone code in specific biological contexts.

Which techniques are validated for Histone H2B type 1-K antibody applications?

Histone H2B type 1-K recombinant monoclonal antibodies have been validated for several experimental applications, including:

• Western Blotting (WB): Recommended at 1:5000-1:10000 dilution for detecting the 14 kDa H2B type 1-K protein in various tissues .

• Immunohistochemistry (IHC): Effective at 1:50-1:500 dilution, particularly for paraffin-embedded human tissue sections .

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated for quantitative detection of H2B type 1-K .

Additional techniques that may be compatible based on related H2B antibody applications include:

• ChIP and ChIP-seq for studying genomic distribution

• Immunofluorescence (IF) for subcellular localization studies

• Dot blot for rapid screening of samples

When designing experiments, researchers should perform validation steps specific to their sample types and experimental conditions to ensure optimal performance of the antibody.

What is the recommended protocol for detecting Histone H2B type 1-K in senescent cells?

For detecting H2B type 1-K accumulation in senescent cells, a combined approach using immunofluorescence and western blotting is recommended:

Immunofluorescence Protocol:

• Culture cells on coverslips and induce senescence using appropriate stimuli (e.g., etoposide for DNA damage-induced senescence)

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 3% BSA in PBS for 1 hour

• Incubate with anti-H2B type 1-K primary antibody (1:100 dilution) overnight at 4°C

• Wash three times with PBS

• Incubate with fluorescently-labeled secondary antibody for 1 hour at room temperature

• Counterstain with DAPI to visualize nuclei

• Mount and image using confocal microscopy

Western Blot Protocol:

• Extract histones using acid extraction protocol (optimized for histone isolation)

• Resolve proteins on a 15% SDS-PAGE gel

• Transfer to PVDF membrane

• Block with 5% non-fat milk in TBST

• Incubate with anti-H2B type 1-K antibody (1:5000 dilution)

• Wash and incubate with HRP-conjugated secondary antibody

• Develop using ECL detection system

For validation, co-staining with senescence markers (e.g., γH2AX for DNA damage, SA-β-Gal for senescence) is recommended to confirm the correlation between H2B type 1-K accumulation and senescent phenotype .

How can mass spectrometry be integrated with antibody-based detection of H2B type 1-K?

Mass spectrometry provides a powerful complementary approach to antibody-based detection of H2B type 1-K, allowing for precise identification and quantification of this histone variant and its post-translational modifications:

Integrated Workflow:

• Immunoprecipitation with H2B type 1-K antibody:

  • Perform chromatin immunoprecipitation using the H2B type 1-K antibody

  • Elute bound proteins for subsequent mass spectrometry analysis

• Mass Spectrometry Analysis:

  • Digest immunoprecipitated samples with trypsin

  • Analyze using UHPLC-MS with high-resolution Orbitrap instrumentation

  • Identify H2B type 1-K by monitoring its specific C-terminal peptide (Leu100-Lys125)

  • Quantify relative abundance compared to other H2B variants

• Validation by Targeted MS:

  • Develop selected reaction monitoring (SRM) assays for H2B type 1-K-specific peptides

  • Use heavy isotope-labeled peptide standards for absolute quantification

This integrated approach provides several advantages over antibody-based methods alone, including the ability to distinguish between highly similar H2B variants and simultaneously analyze post-translational modifications. Research has shown that the relative quantification of the abundance of tryptic C-terminal peptides can unambiguously identify H2B type 1-K and track its accumulation in different biological states .

What are the key considerations when selecting between polyclonal and recombinant monoclonal antibodies for H2B research?

When investigating H2B variants, the choice between polyclonal and recombinant monoclonal antibodies significantly impacts experimental outcomes:

Recombinant rabbit monoclonal antibodies offer several advantages, including better specificity and sensitivity, lot-to-lot consistency, animal origin-free formulations, and broader immunoreactivity due to the larger rabbit immune repertoire . These characteristics make recombinant monoclonal antibodies particularly valuable for discriminating between highly similar H2B variants like H2B type 1-K.

For studies focusing specifically on H2B type 1-K accumulation in senescent cells, recombinant monoclonal antibodies are recommended due to their superior specificity in distinguishing this variant from other H2B family members .

What controls should be included when validating H2B type 1-K antibody specificity?

Proper validation of H2B type 1-K antibody specificity is crucial for reliable research outcomes. The following controls should be incorporated:

Essential Controls:

• Positive Controls:

  • Human testis tissue, which naturally expresses H2B type 1-K

  • Senescent fibroblasts with persistent DNA damage (e.g., etoposide-treated)

  • Recombinant H2B type 1-K protein or synthetic peptide standards

• Negative Controls:

  • Matched isotype control antibody

  • Peptide competition assay using the immunizing peptide

  • Samples known to lack H2B type 1-K expression (e.g., certain quiescent cells)

• Specificity Controls:

  • Cross-reactivity testing against other H2B variants

  • Knockdown/knockout validation using siRNA or CRISPR against HIST1H2BK

  • Mass spectrometry confirmation of immunoprecipitated proteins

Validation Protocol Example:

• Perform western blot with the antibody on human cell lysates

• Include recombinant H2B type 1-K as positive control

• Include other recombinant H2B variants to assess cross-reactivity

• Perform peptide competition assay by pre-incubating antibody with excess immunizing peptide

• Compare staining patterns in senescent vs. non-senescent cells

Rigorous validation using these controls ensures that experimental results accurately reflect the expression and distribution of H2B type 1-K rather than non-specific binding or cross-reactivity with other histone variants.

How can researchers address variability in H2B type 1-K antibody performance across different experimental systems?

Variability in antibody performance can significantly impact experimental reproducibility. Researchers can implement the following strategies to address this challenge:

• Standardize Sample Preparation:

  • For histones, use acid extraction methods consistently (e.g., 0.2N HCl extraction)

  • Control fixation conditions rigorously for IHC and IF (fixation time, temperature)

  • Standardize chromatin preparation protocols for ChIP applications

• Optimize Antibody Conditions:

  • Perform titration experiments to determine optimal antibody concentration

  • Test multiple blocking agents (BSA, milk, serum) to reduce background

  • Evaluate different incubation temperatures and times

  • For IHC, compare antigen retrieval methods (citrate buffer pH 6.0 has shown good results)

• Quantitative Validation:

  • Use ChIP-qPCR with known target regions to assess antibody performance

  • Implement spike-in controls for normalization

  • Use densitometry analysis for western blots with reference standards

• Cross-Platform Validation:

  • Confirm H2B type 1-K detection using complementary techniques

  • Correlate antibody-based detection with mass spectrometry data

  • Compare results across different cell types and experimental conditions

When working with senescent cells, researchers should particularly note that H2B type 1-K accumulation is specific to deep senescent conditions with persistent DNA damage and might not be detectable in other senescent states . Accordingly, experimental systems should be carefully characterized for senescence markers and DNA damage indicators (e.g., γH2AX foci) to correctly interpret H2B type 1-K antibody results.

How can H2B type 1-K antibodies be leveraged for studying chromatin changes during cellular aging?

H2B type 1-K antibodies offer unique opportunities for investigating chromatin alterations associated with cellular aging:

• Senescence-Associated Chromatin Mapping:

  • Use ChIP-seq with H2B type 1-K antibodies to map genome-wide distribution in young versus senescent cells

  • Identify genomic regions enriched for H2B type 1-K in senescent cells

  • Correlate with transcriptional changes and other epigenetic marks

• Multiplex Imaging Approaches:

  • Combine H2B type 1-K immunofluorescence with other senescence markers (p16, p21, γH2AX)

  • Implement multiplex imaging to simultaneously visualize H2B type 1-K with chromatin compaction markers

  • Quantify spatial relationships between H2B type 1-K enrichment and senescence-associated heterochromatin foci (SAHF)

• In vivo Detection of Senescent Cells:

  • Develop H2B type 1-K-based methods to identify senescent cells in tissue sections

  • Compare H2B type 1-K accumulation patterns across different aged tissues

  • Correlate with age-related pathologies and interventions

Research has demonstrated that H2B type 1-K accumulation is a specific feature of deep senescent cells with persistent DNA damage . This unique characteristic can be exploited to develop more precise methods for identifying and studying specific subpopulations of senescent cells in aging tissues, potentially leading to better understanding of the heterogeneity of cellular aging processes.

What is the potential for using H2B type 1-K as a biomarker in clinical research?

The specific accumulation of H2B type 1-K in senescent cells with persistent DNA damage suggests promising applications as a biomarker in clinical research:

• Aging and Age-Related Diseases:

  • Quantify H2B type 1-K-positive cells in tissues from patients of different ages

  • Correlate with markers of biological aging and age-related pathologies

  • Evaluate changes in H2B type 1-K patterns in response to anti-aging interventions

• Cancer Research Applications:

  • Assess therapy-induced senescence using H2B type 1-K as a marker

  • Distinguish between different senescent states in tumor microenvironments

  • Monitor senescence induction during chemotherapy and radiotherapy

• Methodological Considerations for Clinical Samples:

  • Optimize tissue preservation and processing for H2B type 1-K detection

  • Develop quantitative image analysis workflows for clinical specimens

  • Integrate with other clinical biomarkers for comprehensive assessment

The combination of H2B type 1-K with other senescence markers may provide a more accurate identification of senescent cells in clinical samples. For instance, the co-detection of H2B type 1-K with HMGA1 modifications, which also accumulate exclusively in cells with persistent DNA damage , could improve the specificity of senescent cell identification in patient tissues.