HIST1H1B (Ab-137) Antibody

What is the HIST1H1B (Ab-137) Antibody and what cellular processes does it help investigate?

The HIST1H1B (Ab-137) Antibody (PACO56611) is a rabbit-derived polyclonal antibody that specifically targets the human histone H1B protein at the threonine-137 site. This antibody enables researchers to investigate histone H1B's critical role in chromatin organization and gene expression regulation. Histone H1B functions as a linker histone that binds to nucleosomes and stabilizes higher-order chromatin structures . Unlike core histones (H2A, H2B, H3, H4), linker histones exhibit more dynamic association with chromatin, making them particularly important for studying transient and developmentally regulated chromatin condensation processes .

Research using this antibody provides insights into epigenetic regulation mechanisms, as histone H1B influences DNA packaging and accessibility to transcriptional machinery . The antibody allows detection and analysis of this important histone protein across multiple experimental approaches, making it valuable for epigenetics, gene regulation, and chromatin structure investigations.

What validated applications can the HIST1H1B (Ab-137) Antibody be used for?

The HIST1H1B (Ab-137) Antibody has been validated for multiple research applications:

• Western Blotting (WB): Successfully detects HIST1H1B in multiple human cell lines including U87, K562, and HEK293 whole cell lysates, with recommended dilution ranges of 1:100-1:1000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Effective at dilutions between 1:2000-1:10000, allowing quantitative analysis of HIST1H1B proteins .

• Immunohistochemistry (IHC): Functions at dilutions between 1:20-1:200 for detecting HIST1H1B in tissue sections .

These multiple applications make the antibody versatile for complementary experimental approaches, enabling researchers to validate findings across different methodologies.

What cell lines have been validated for HIST1H1B (Ab-137) Antibody detection?

The HIST1H1B (Ab-137) Antibody has demonstrated reliable detection in the following human cell lines:

For optimal results when using Western blotting with these cell lines, researchers should pair the primary antibody (HIST1H1B antibody at 1μg/ml) with an appropriate secondary antibody, such as goat polyclonal to rabbit IgG at 1/50000 dilution .

How should experimental controls be designed when using HIST1H1B (Ab-137) Antibody in chromatin studies?

When designing experiments with the HIST1H1B (Ab-137) Antibody, researchers should implement several critical controls:

• Negative Controls:

  • Isotype control (rabbit IgG) at equivalent concentration to evaluate non-specific binding

  • Samples from cell lines with HIST1H1B knockdown/knockout (if available)

  • Secondary antibody-only controls to assess background signal

• Positive Controls:

  • Cell lines with confirmed HIST1H1B expression (U87, K562, HEK293)

  • Recombinant HIST1H1B protein (if available)

  • Comparison with other validated anti-HIST1H1B antibodies targeting different epitopes

• Cross-Reactivity Controls:

  • Assessment of signal in non-human samples (antibody is specifically reactive with human samples)

  • Evaluation of potential cross-reactivity with other H1 histone family members

• Blocking Peptide Controls:

  • Pre-incubation of the antibody with the immunizing peptide (sequence around Thr-137) to confirm specificity

These controls are particularly important given the high sequence homology between histone variants and the potential for cross-reactivity in histone studies.

How can researchers investigate the relationship between HIST1H1B and R-loop formation in genomic instability studies?

Investigating HIST1H1B's role in preventing R-loop formation and genomic instability requires a multi-faceted experimental approach:

• HIST1H1B Depletion Studies:

  • Generate HIST1H1B knockdown cell lines using siRNA or CRISPR-Cas9

  • Quantify R-loop formation using S9.6 antibody (specific for DNA:RNA hybrids) in immunofluorescence or DNA:RNA immunoprecipitation (DRIP) assays

  • Measure γH2Av (H2AvS137P) content as a marker of DNA damage, particularly in heterochromatic regions

• Cell Cycle Analysis:

  • Synchronize cells and assess R-loop accumulation in G1-phase

  • Measure DNA damage markers (γH2Av) during DNA replication phase

  • Use flow cytometry to correlate HIST1H1B levels with cell cycle stages

• Genomic Instability Assessment:

  • Quantify sister chromatid exchanges and DNA breaks in HIST1H1B-depleted cells

  • Analyze JNK-mediated apoptosis pathways that may be activated

• Rescue Experiments:

  • Reintroduce wild-type HIST1H1B to depleted cells and measure reversal of R-loop formation

  • Test HIST1H1B mutants (especially at Thr-137) to identify critical residues for R-loop prevention

This experimental framework leverages findings that histone H1 prevents R-loop-induced DNA damage in heterochromatin, suggesting a crucial role for HIST1H1B in maintaining genome stability .

How is HIST1H1B implicated in lymphoid malignancies and what research methods can elucidate these connections?

HIST1H1B and other H1 family members have been implicated in various lymphoid malignancies, with specific methodological approaches required to investigate these connections:

• Mutation Analysis in Lymphoma Subtypes:

  • Sequence HIST1H1B in patient samples from diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, and follicular lymphoma

  • Focus particularly on missense mutations that affect the globular domain (GD) and C-terminal domain

  • Correlate mutations with reduced chromatin association or compaction capabilities

• Functional Assessment of Mutant HIST1H1B:

  • Express wild-type and mutant forms in lymphoma cell lines

  • Measure chromatin compaction using accessibility assays (ATAC-seq, DNase-seq)

  • Assess distribution of core histone modifications that may be altered by HIST1H1B mutations

  • Analyze reactivation of stem cell-specific transcriptional programs

• HIST1H1B in Lymphoma Classification:

  • Evaluate HIST1H1B mutation status in relation to established lymphoma subtypes, particularly MCD-DLBCLs and ABC-DLBCLs

  • Correlate with clinical outcomes to determine prognostic value

• Epigenetic Interplay Analysis:

  • Investigate interactions between HIST1H1B and Polycomb group proteins

  • Study H1-dependent stimulation of PRC2 function and resulting H3K27 methylation patterns

  • Analyze how HIST1H1B mutations affect H2A K119 ubiquitylation by variant PRC1 complexes

These approaches can help elucidate how HIST1H1B mutations contribute to aberrant chromatin architecture and transcriptional regulation in lymphoid malignancies.

What methodological approaches can determine if HIST1H1B (Ab-137) Antibody is suitable for detecting mutated forms of the protein in lymphoma samples?

To determine if the HIST1H1B (Ab-137) Antibody can detect mutated forms of the protein in lymphoma samples:

• Epitope Mapping and Mutation Database Analysis:

  • Precisely map the antibody's recognition site around Thr-137

  • Cross-reference with known mutation hotspots in lymphoma to identify potential epitope disruptions

  • Perform in silico prediction of antibody binding to common HIST1H1B mutants

• Recombinant Protein Testing:

  • Generate recombinant HIST1H1B proteins with known lymphoma-associated mutations

  • Perform Western blotting to assess antibody binding to wild-type versus mutant proteins

  • Quantify binding affinities for different mutant forms

• Patient Sample Validation:

  • Perform immunohistochemistry on lymphoma tissue microarrays with known HIST1H1B mutation status

  • Compare antibody signal between wild-type and mutant samples

  • Correlate with parallel detection methods (e.g., RNA-seq for expression, DNA sequencing for mutation confirmation)

• Functional Validation:

  • Assess whether the antibody can detect HIST1H1B with altered chromatin association

  • Compare nuclear vs. cytoplasmic fractions in cells expressing mutant HIST1H1B

  • Evaluate co-localization with heterochromatin markers in wild-type vs. mutant contexts

These methodological steps will determine whether the antibody's epitope (around Thr-137) remains accessible and recognizable in mutated forms of HIST1H1B associated with lymphoid malignancies.

How can the HIST1H1B (Ab-137) Antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

While ChIP is not listed among the validated applications for this specific antibody, researchers may optimize it for this purpose through the following protocol adaptations:

• Cross-linking Optimization:

  • Test multiple cross-linking conditions (formaldehyde concentrations from 0.5-2%, incubation times of 5-15 minutes)

  • Consider dual cross-linking with ethylene glycol bis(succinimidyl succinate) (EGS) followed by formaldehyde to better preserve protein-protein interactions involving linker histones

• Chromatin Fragmentation:

  • Optimize sonication conditions to generate fragments of 200-500bp

  • Verify fragmentation efficiency using gel electrophoresis before proceeding

• Antibody Titration and Validation:

  • Test multiple antibody concentrations (starting with manufacturer's recommendations for other applications)

  • Include positive controls (regions known to be enriched for H1 binding) and negative controls (regions typically depleted of H1)

  • Verify specificity using competing peptide controls or HIST1H1B knockout/knockdown samples

• Buffer Optimization:

  • Test various detergent concentrations to reduce background while maintaining specific interactions

  • Adjust salt concentrations to optimize antibody-antigen binding

• Sequential ChIP Considerations:

  • For studies of HIST1H1B co-localization with other chromatin marks, optimize sequential ChIP protocols

  • Test whether HIST1H1B (Ab-137) Antibody works better in the first or second immunoprecipitation round

What methodological approaches can resolve contradictory results between HIST1H1B detection using Ab-137 versus other epitope-targeting antibodies?

When facing contradictory results between antibodies targeting different HIST1H1B epitopes:

• Epitope Accessibility Analysis:

  • Conduct epitope mapping to determine if the Thr-137 region might be masked under certain experimental conditions

  • Compare with accessibility of alternative epitopes (such as Lys-66 targeted by PACO56632)

  • Test denaturation conditions that may differentially affect epitope exposure

• Isoform Specificity Verification:

  • Perform immunoprecipitation followed by mass spectrometry to confirm which specific H1 variants are being detected

  • Use recombinant expression of specific H1 variants as positive controls

  • Employ isoform-specific knockdown to validate antibody specificity

• Post-translational Modification Interference:

  • Investigate whether post-translational modifications near Thr-137 affect Ab-137 binding

  • Use phosphatase treatment to remove potential phosphorylation at Thr-137 that might interfere with antibody recognition

  • Compare results in cell types with different known PTM profiles for HIST1H1B

• Methodological Cross-validation:

  • Apply multiple detection methods (Western blot, immunofluorescence, flow cytometry)

  • Standardize protocols and sample preparation across antibodies

  • Quantify relative signals and establish conversion factors between antibodies

• Combined Antibody Approach:

  • Use both antibodies simultaneously in multiplexed detection

  • Identify conditions where signals correlate versus diverge

  • Develop consensus reporting that acknowledges epitope-specific differences

How can researchers utilize the HIST1H1B (Ab-137) Antibody to investigate the role of linker histones in three-dimensional chromatin architecture?

Investigating three-dimensional chromatin architecture using the HIST1H1B (Ab-137) Antibody involves integrating multiple advanced methodologies:

• Chromosome Conformation Capture Technologies:

  • Combine ChIP using HIST1H1B (Ab-137) Antibody with Hi-C or Micro-C (ChIP-Hi-C)

  • Identify HIST1H1B-mediated long-range chromatin interactions

  • Compare chromatin interaction maps before and after HIST1H1B depletion

• Super-resolution Microscopy:

  • Perform immunofluorescence with HIST1H1B (Ab-137) Antibody using techniques like STORM or PALM

  • Analyze co-localization with architectural proteins (CTCF, cohesin)

  • Measure changes in chromatin compaction at the nanoscale level

• Polymer Physics Modeling:

  • Use HIST1H1B binding data to inform computational models of chromatin folding

  • Simulate the effect of HIST1H1B depletion or mutation on predicted 3D structures

  • Validate computational predictions with experimental data

• Nuclear Architecture Analysis:

  • Investigate HIST1H1B distribution relative to nuclear landmarks (lamina, nucleolus)

  • Assess changes in heterochromatin organization upon HIST1H1B mutation or depletion

  • Correlate with changes in gene expression in specific nuclear compartments

This research direction is particularly relevant given the emerging role of aberrant three-dimensional chromatin architecture in malignancy and the capacity of HIST1H1B to regulate chromatin compaction and higher-order structures .

What methodological frameworks can determine whether HIST1H1B isoform-specific functions observed in lymphoma are relevant in other cancer types?

To investigate whether HIST1H1B functions are cancer-type specific:

• Multi-cancer Analysis Pipeline:

  • Perform comprehensive mutation screening of HIST1H1B across cancer databases

  • Conduct immunohistochemistry with HIST1H1B (Ab-137) Antibody on tissue microarrays spanning multiple cancer types

  • Correlate HIST1H1B expression/mutation patterns with cancer subtypes and clinical outcomes

• Functional Comparisons Across Cell Types:

  • Deplete HIST1H1B in cell line panels representing diverse cancer types

  • Compare effects on chromatin compaction, R-loop formation, and genomic stability

  • Assess cancer-type specificity of transcriptional changes following HIST1H1B manipulation

• Isoform Substitution Experiments:

  • Perform rescue experiments with different H1 isoforms in HIST1H1B-depleted cells from various cancer types

  • Determine whether functional interchangeability of H1 isoforms is universal or context-dependent

  • Identify cancer types where HIST1H1B has unique, non-redundant functions

• Epigenetic Context Mapping:

  • Profile the distribution of histone modifications across cancer types

  • Analyze whether HIST1H1B binding correlates with different epigenetic signatures in different cancers

  • Investigate interactions between HIST1H1B and cancer-specific chromatin remodelers

This methodological framework can help determine if the HIST1H1B functions observed in lymphoma represent a general mechanism in cancer biology or a lymphoid-specific phenomenon.