Formyl-HIST1H2BC (K108) Antibody

What is Formyl-Histone H2B (K108) and why is it significant in epigenetic research?

Formyl-Histone H2B (K108) refers to a specific post-translational modification of the histone H2B protein, where the lysine residue at position 108 is formylated. This modification plays a critical role in gene regulation, chromatin structure organization, and DNA repair processes. As a core component of nucleosomes, histone H2B helps wrap and compact DNA into chromatin, thereby limiting DNA accessibility to cellular machineries that require DNA as a template . The formylation at K108 represents one of many post-translational modifications that constitute the "histone code," which regulates DNA accessibility and influences transcription regulation, DNA repair, DNA replication, and chromosomal stability . Research into this specific modification helps uncover mechanisms of epigenetic regulation and may reveal new therapeutic targets for diseases associated with aberrant histone modifications, including cancer and developmental disorders .

How does Formyl-Histone H2B (K108) differ from other histone modifications?

Formyl-Histone H2B (K108) represents a specific type of histone modification that differs from other modifications in several key aspects. While many histone modifications involve methylation, acetylation, or phosphorylation, formylation is a distinct chemical modification that likely serves unique regulatory functions. For example, the 2-hydroxyisobutyryl modification at the same position (K108) represents a different type of modification with potentially distinct functions in regulating gene expression and chromatin structure . Each type of histone modification creates a specific recognition site for effector proteins that mediate downstream biological processes. Formyl-Histone H2B (K108) may interact with specific reader proteins that recognize this modification to influence gene expression patterns or chromatin accessibility in ways that differ from other histone marks . Understanding these differences is crucial for mapping the complete histone code and its biological significance.

Which biological systems and cell types express detectable levels of Formyl-Histone H2B (K108)?

Formyl-Histone H2B (K108) has been detected in various biological systems and cell types. Based on available immunohistochemistry data, this modification is present in:

• Human tissues: liver, colon, and cervical epithelial cells (HeLa cells)

• Mouse tissues: liver and lung, as well as embryonic fibroblast cells (NIH/3T3)

• Rat tissues: brain and colon

The nuclear localization of this modification has been confirmed through immunohistochemical analysis, with specific staining observed in the nucleus of cells from these tissues . The wide distribution across different species and tissue types suggests that this histone modification may serve fundamental biological functions that are conserved across mammals. Researchers should note that expression levels may vary depending on cell type, developmental stage, and physiological conditions, making it important to validate detection in specific experimental systems .

What are the validated applications for Formyl-Histone H2B (K108) antibodies in epigenetic research?

Formyl-Histone H2B (K108) antibodies have been validated for multiple experimental applications in epigenetic research. The following applications have been confirmed through rigorous testing:

• Western Blotting (WB): Successfully applied at dilutions of 1:2000 for detecting the modified histone in human and mouse cell lysates, with observed bands at the expected size of 14 kDa

• Immunohistochemistry-Paraffin (IHC-P): Validated at dilutions between 1:50-1:800 for detecting nuclear staining in human, mouse, and rat tissue sections

• Peptide Array (PepArr): Confirmed high specificity through testing against 501 different modified and unmodified histone peptides, demonstrating selective binding to the target modification

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated for detecting the modified protein in solution-based assays

• Immunofluorescence and Immunoprecipitation: Useful for visualizing cellular localization and protein interactions

• Chromatin Immunoprecipitation (ChIP): Valuable for studying genomic distribution of the modification and its association with regulatory elements

Researchers should note that optimal dilutions and conditions may vary depending on sample type and experimental setup, with recommended starting dilutions provided for each application (e.g., 1:50-1:200 for IHC-P) .

What antigen retrieval methods are recommended for immunohistochemical detection of Formyl-Histone H2B (K108)?

For optimal immunohistochemical detection of Formyl-Histone H2B (K108) in formalin-fixed, paraffin-embedded (FFPE) tissues, specific antigen retrieval methods have proven effective:

• Heat-mediated antigen retrieval using Tris/EDTA buffer at pH 9.0 has been validated for human, mouse, and rat tissues prior to IHC staining protocols . This method effectively reverses formalin-induced protein cross-links that may mask the epitope.

• High-pressure antigen retrieval performed with 0.01M Citrate Buffer (pH 6.0) has also been successfully employed prior to IHC staining, particularly for human liver tissue samples .

The choice between these methods may depend on specific tissue types and fixation conditions. For optimal results, researchers should:

• Ensure complete coverage of tissue sections with retrieval buffer

• Maintain consistent heating conditions throughout the retrieval process

• Allow sections to cool gradually to room temperature before proceeding with blocking steps

• Validate the retrieval method by comparing staining intensity and specificity with positive controls

Following antigen retrieval, a blocking step using 5% non-fat dry milk (NFDM) in TBST has been shown to minimize background staining and improve signal-to-noise ratio .

How should samples be prepared for Western blot analysis of Formyl-Histone H2B (K108)?

Effective sample preparation for Western blot analysis of Formyl-Histone H2B (K108) requires specific protocols to preserve the histone modification and ensure optimal detection:

• Cell Lysis and Histone Extraction:

  • Use specialized histone extraction buffers containing HDAC inhibitors (such as sodium butyrate) and protease inhibitors to preserve modifications

  • For total cell lysates, apply 10 μg of protein per lane as validated with HeLa and NIH/3T3 cell samples

  • Consider acid extraction methods (e.g., 0.2N HCl) for enrichment of histones from nuclear fractions

• Gel Electrophoresis and Transfer:

  • Use 15-18% SDS-PAGE gels to effectively resolve the low molecular weight histone proteins

  • Transfer to PVDF membranes at lower voltage (e.g., 30V overnight) to prevent small proteins from passing through the membrane

  • Consider wet transfer methods rather than semi-dry for more consistent results with histone proteins

• Blocking and Antibody Incubation:

  • Block membranes in 5% non-fat dry milk (NFDM) in TBST buffer

  • Apply Formyl-Histone H2B (K108) antibody at a 1:2000 dilution in blocking buffer

  • For secondary detection, anti-rabbit IgG-HRP can be used at 1:50,000 dilution

• Expected Results:

  • The predicted band size for Histone H2B is 14 kDa

  • Observed band size in validated experiments is 14 kDa

  • Short exposure times (10-15 seconds) may be sufficient for detection depending on expression levels

This optimized protocol has been validated using human (HeLa) and mouse (NIH/3T3) cell lysates, demonstrating consistent and specific detection of the modified histone .

How can ChIP-seq be optimized for genome-wide mapping of Formyl-Histone H2B (K108)?

Optimizing ChIP-seq for genome-wide mapping of Formyl-Histone H2B (K108) requires careful attention to several methodological aspects:

• Crosslinking and Chromatin Preparation:

  • Use dual crosslinking with both formaldehyde (1% for 10 minutes) and protein-specific crosslinkers like disuccinimidyl glutarate (DSG) to improve capture of histone-DNA interactions

  • Optimize sonication conditions to achieve chromatin fragments of 200-500 bp, which is ideal for high-resolution mapping

  • Include spike-in controls with chromatin from different species to allow normalization across experiments

• Immunoprecipitation Protocol:

  • Pre-clear chromatin with protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of Formyl-Histone H2B (K108) antibody per ChIP reaction, which has been validated for high specificity in peptide array assays

  • Extend incubation times (overnight at 4°C) to improve capture of the modified histones

  • Include appropriate negative controls (IgG) and positive controls (antibodies against abundant histone marks)

• Library Preparation and Sequencing:

  • Use library preparation methods optimized for low-input samples if ChIP yield is limited

  • Perform paired-end sequencing at sufficient depth (30-50 million reads) to ensure comprehensive coverage

  • Consider using unique molecular identifiers (UMIs) to control for PCR duplication artifacts

• Data Analysis Considerations:

  • Apply peak calling algorithms optimized for histone modifications (e.g., MACS2 with broad peak settings)

  • Analyze correlation with other histone marks and transcriptional activity to understand functional significance

  • Integrate with RNA-seq data to correlate modification patterns with gene expression changes

This approach has been successfully applied for similar histone modifications and can be adapted specifically for Formyl-Histone H2B (K108) to map its genomic distribution and potential regulatory functions .

What is the relationship between Formyl-Histone H2B (K108) and other histone modifications in the context of gene regulation?

The relationship between Formyl-Histone H2B (K108) and other histone modifications reveals complex interaction patterns within the histone code that influence gene regulation:

• Co-occurrence Patterns:

  • Formyl-Histone H2B (K108) may co-exist with specific histone marks associated with active transcription, such as H3K4me3 or H3K27ac, in regulatory regions

  • The combination of Formyl-Histone H2B (K108) with other modifications likely creates unique recognition surfaces for effector proteins

  • Sequential ChIP experiments (Re-ChIP) can be employed to determine co-occurrence of multiple modifications on the same nucleosome

• Cross-talk Mechanisms:

  • The presence of Formyl-Histone H2B (K108) may influence the deposition or removal of other modifications through writer/eraser enzyme recruitment

  • Other modifications on the same nucleosome can potentially alter the accessibility of K108 to formylation enzymes

  • Studies investigating competition between different modifications (e.g., formylation versus 2-hydroxyisobutyrylation) at the K108 position would reveal potential regulatory switches

• Impact on Chromatin Structure:

  • As a core component of nucleosomes, modified H2B influences DNA accessibility to transcription machinery

  • The formylation at K108 likely affects nucleosome stability or positioning, potentially altering higher-order chromatin structure

  • The modification may create recognition sites for ATP-dependent chromatin remodeling complexes

• Disease Contexts:

  • Aberrant patterns of Formyl-Histone H2B (K108) in relation to other histone marks have been implicated in cancer and developmental disorders

  • The balance between different modifications at K108 (formylation vs. other modifications) may be disturbed in disease states

  • Therapeutic approaches targeting writers, readers, or erasers of these modifications represent potential intervention strategies

Understanding these relationships requires integrated epigenomic approaches combining ChIP-seq for multiple histone marks with transcriptome and chromatin accessibility analyses .

How does Formyl-Histone H2B (K108) contribute to antimicrobial functions of histones?

Histone H2B has been recognized for its broad antimicrobial activity, and the Formyl-K108 modification may play a specific role in this function:

• Antimicrobial Mechanism:

  • Histone H2B contributes to the formation of functional antimicrobial barriers in the colonic epithelium and participates in the bactericidal activity of amniotic fluid

  • The positive charge of histones facilitates interaction with negatively charged bacterial membranes, leading to membrane disruption

  • Formylation at K108 may alter the local charge distribution, potentially enhancing or modulating the antimicrobial properties of H2B fragments

• Tissue-Specific Expression:

  • Immunohistochemical analysis has confirmed the presence of Formyl-Histone H2B (K108) in tissues with significant antimicrobial defense requirements, including:

    • Human colon tissue

    • Mouse liver tissue

    • Rat colon tissue

  • The nuclear localization of this modification suggests it may serve as a reservoir for antimicrobial histone fragments upon cellular stress or damage

• Connection to Innate Immunity:

  • During infection or inflammation, histone modifications like Formyl-K108 may signal for specific immune responses

  • Modified histones released during cell death (NETosis, necrosis) can act as damage-associated molecular patterns (DAMPs)

  • The formyl group could potentially be recognized by formyl peptide receptors on immune cells, similar to bacterial formylated peptides

• Research Approaches:

  • Antimicrobial assays comparing wild-type H2B with K108 mutants (K108A or K108R) can elucidate the specific contribution of this residue

  • Mass spectrometry analysis of histone fragments released during infection can identify if K108-formylated peptides have enhanced antimicrobial activity

  • In vivo models with modified expression of histone modifying enzymes can assess the physiological relevance of this modification in host defense

This dual role of H2B as both a chromatin component and antimicrobial peptide precursor represents an intriguing connection between epigenetic regulation and innate immunity .

What are common sources of non-specific binding when using Formyl-Histone H2B (K108) antibodies, and how can they be mitigated?

Non-specific binding can compromise experimental results when using Formyl-Histone H2B (K108) antibodies. Common sources and mitigation strategies include:

• Cross-reactivity with Similar Epitopes:

  • Challenge: The antibody may recognize similar formylated lysine residues on other histones or proteins

  • Solution: Validate antibody specificity using peptide arrays with multiple modified and unmodified histone peptides

  • Implementation: Use antibodies that have been tested against 501 different histone peptides, as demonstrated in peptide array validation experiments

• Insufficient Blocking:

  • Challenge: Inadequate blocking leads to high background signal in Western blots and IHC

  • Solution: Optimize blocking conditions using 5% non-fat dry milk (NFDM) in TBST buffer

  • Implementation: Extend blocking time to 1-2 hours at room temperature and ensure complete coverage of membranes/slides

• Overfixation Effects in IHC:

  • Challenge: Excessive formalin fixation can mask epitopes and increase non-specific binding

  • Solution: Perform heat-mediated antigen retrieval with Tris/EDTA buffer (pH 9.0) or citrate buffer (pH 6.0)

  • Implementation: Optimize retrieval time and temperature for each tissue type

• Secondary Antibody Cross-reactivity:

  • Challenge: Secondary antibodies may bind to endogenous immunoglobulins in tissue samples

  • Solution: Include secondary-only control in all experiments (using PBS instead of primary antibody)

  • Implementation: Compare experimental results with secondary-only controls to identify true positive signals

• Endogenous Peroxidase Activity:

  • Challenge: Endogenous peroxidase can cause false positive signals in IHC using HRP-conjugated detection systems

  • Solution: Include hydrogen peroxide quenching step (0.3% H₂O₂ in methanol) before primary antibody incubation

  • Implementation: Apply quenching solution for 10-15 minutes followed by thorough washing

By implementing these strategies, researchers can significantly improve signal specificity and data reliability when working with Formyl-Histone H2B (K108) antibodies .

How can the specificity of Formyl-Histone H2B (K108) antibody be validated in different experimental systems?

Validating the specificity of Formyl-Histone H2B (K108) antibody across different experimental systems requires multiple complementary approaches:

• Peptide Competition Assays:

  • Method: Pre-incubate antibody with synthetic peptides containing formylated K108 before application to samples

  • Expected Result: Specific binding should be blocked by the formylated peptide but not by unmodified or differently modified peptides

  • Implementation: Use peptides at concentrations of 0.1-10 μg/ml in antibody dilution solution

• Peptide Array Analysis:

  • Method: Test antibody binding against multiple modified and unmodified histone peptides printed at different concentrations

  • Expected Result: Strong and selective binding to formyl-K108 peptides as demonstrated in validated arrays testing 501 different histone peptides

  • Analysis: Calculate binding affinity as area under curve when antibody binding values are plotted against peptide concentration

• Genetic Validation Approaches:

  • Method: Compare antibody signal in wild-type cells versus cells with K108R mutation (preventing modification) or knockout of enzymes responsible for formylation

  • Expected Result: Decreased or absent signal in mutant/knockout systems

  • Implementation: Use CRISPR-Cas9 genome editing to generate K108R mutant cell lines for validation

• Mass Spectrometry Correlation:

  • Method: Perform immunoprecipitation with the antibody followed by mass spectrometry analysis of pulled-down proteins

  • Expected Result: Enrichment of peptides containing formylated K108

  • Implementation: Use targeted MS approaches like parallel reaction monitoring (PRM) for sensitive detection

• Cross-species Validation:

  • Method: Test antibody reactivity in tissues from multiple species (human, mouse, rat) with high sequence conservation around K108

  • Expected Result: Consistent nuclear staining pattern across species as observed in validated IHC experiments

  • Implementation: Include positive control tissues known to express the modification (e.g., human liver, mouse lung, rat brain)

This multi-faceted validation approach ensures robust and reliable detection of Formyl-Histone H2B (K108) across diverse experimental conditions and biological systems .

What controls should be included in experiments using Formyl-Histone H2B (K108) antibodies?

A comprehensive set of controls is essential for generating reliable and interpretable data when using Formyl-Histone H2B (K108) antibodies:

• Primary Controls:

  Control Type	Implementation	Purpose	Expected Result
  Negative Control	Secondary antibody only (PBS instead of primary)	Assess non-specific binding of secondary antibody	No signal or minimal background
  Isotype Control	Non-specific IgG from same host species (rabbit)	Evaluate background from primary antibody isotype	No specific nuclear staining
  Peptide Competition	Pre-incubation with synthetic formyl-K108 peptide	Confirm epitope specificity	Significant reduction in signal
  Positive Control	Known positive tissues (human liver, mouse lung, rat brain)	Verify antibody functionality	Clear nuclear staining pattern

• Sample-specific Controls:

  • Include cell lines or tissues with validated high expression of the modification

  • When possible, include samples treated with histone deacetylase inhibitors to preserve modifications

  • For disease studies, pair experimental samples with appropriate normal tissue controls

• Technical Controls:

  • For Western blotting: Include molecular weight markers and loading controls (total H2B or H3)

  • For IHC: Apply standardized staining protocols with consistent incubation times and temperatures

  • For ChIP: Include input control (pre-immunoprecipitation chromatin) and IgG control IP

• Validation Controls:

  • When available, use cells with genetic manipulation of the K108 residue (K108R mutant)

  • Compare results with alternative antibody clones targeting the same modification

  • Correlate antibody-based detection with mass spectrometry analysis

• Dilution Series:

  • Test antibody performance across a range of dilutions (e.g., 1:50 - 1:800 for IHC-P)

  • Determine optimal concentration that maximizes specific signal while minimizing background

Proper implementation of these controls substantially increases confidence in experimental results and facilitates troubleshooting when unexpected results occur .

How can Formyl-Histone H2B (K108) analysis contribute to understanding cancer development and progression?

Formyl-Histone H2B (K108) analysis offers valuable insights into cancer biology through several research avenues:

• Biomarker Development:

  • Altered patterns of histone modifications, including Formyl-Histone H2B (K108), have been linked to various cancers and developmental disorders

  • IHC analysis of tumor tissues using validated antibodies (1:50-1:800 dilution) can reveal aberrant nuclear staining patterns

  • Correlation of modification levels with clinical outcomes may identify prognostic indicators

  • Quantitative analysis across cancer subtypes could reveal modification signatures associated with specific molecular pathways

• Epigenetic Dysregulation Mechanisms:

  • Formyl-Histone H2B (K108) influences chromatin structure and DNA accessibility, potentially affecting oncogene expression or tumor suppressor silencing

  • Changes in this modification may disrupt normal nucleosome dynamics and DNA repair processes

  • Investigation of enzymes responsible for writing or erasing this modification may identify novel therapeutic targets

  • Integrated analysis with other epigenetic marks can reveal cancer-specific epigenetic signatures

• Therapeutic Development:

  • Understanding the role of Formyl-Histone H2B (K108) in gene regulation may uncover new therapeutic targets

  • Compounds targeting writer or eraser enzymes for this modification could be developed as epigenetic therapies

  • Response to existing epigenetic drugs (HDAC inhibitors, etc.) may correlate with Formyl-Histone H2B (K108) status

  • Combination therapies targeting multiple histone modifications could overcome resistance mechanisms

• Experimental Approaches:

  • ChIP-seq analysis comparing normal vs. tumor tissues can map genome-wide changes in modification distribution

  • Mass spectrometry-based quantification can assess global changes in modification levels

  • CRISPR-mediated modulation of K108 status can elucidate functional consequences in cancer models

  • Patient-derived xenografts can be used to study modification dynamics during tumor progression and treatment

This multifaceted approach to studying Formyl-Histone H2B (K108) in cancer contexts may reveal novel diagnostic markers and therapeutic strategies .

What insights can single-cell epigenomics provide about Formyl-Histone H2B (K108) heterogeneity in complex tissues?

Single-cell epigenomic approaches offer unprecedented insights into Formyl-Histone H2B (K108) heterogeneity within complex tissues:

• Cellular Resolution Analysis:

  • Traditional bulk assays mask cell-specific differences in histone modifications

  • Single-cell techniques reveal modification patterns across distinct cell populations within heterogeneous tissues

  • This is particularly valuable for tissues with known Formyl-Histone H2B (K108) expression such as human liver, colon, and brain tissues

  • Cell-type specific patterns may correlate with specialized functions or developmental states

• Methodological Approaches:

  • Single-cell CUT&Tag: Adapts Cleavage Under Targets and Tagmentation for low cell numbers to map histone modifications

  • scChIC-seq: Single-cell chromatin immunocleavage sequencing using antibodies against Formyl-Histone H2B (K108)

  • Mass cytometry (CyTOF) with metal-conjugated histone modification antibodies for quantitative single-cell profiling

  • Imaging-based approaches: Multiplexed immunofluorescence with highly specific antibodies (e.g., 1:50-1:200 dilution)

• Integration with Other Omics Data:

  • Combined analysis with single-cell RNA-seq to correlate modification patterns with gene expression

  • Multi-omics approaches linking epigenetic profiles to chromatin accessibility and protein expression

  • Trajectory analysis to map modification changes during cellular differentiation or disease progression

  • Spatial transcriptomics integration to preserve tissue context while analyzing modification patterns

• Biological Insights:

  • Identification of rare cell populations with unique modification signatures

  • Mapping of epigenetic heterogeneity in normal tissues versus disease states

  • Understanding how environmental factors influence modification patterns at single-cell resolution

  • Elucidating the dynamics of histone modification during cellular responses to stimuli

This single-cell perspective provides a more nuanced understanding of how Formyl-Histone H2B (K108) contributes to cellular identity and function within complex tissues, potentially revealing cell-type specific regulatory mechanisms that would be missed in bulk analyses .

How might Formyl-Histone H2B (K108) research inform development of epigenetic therapies?

Research on Formyl-Histone H2B (K108) has significant potential to inform the development of next-generation epigenetic therapies:

• Target Identification and Validation:

  • Identification of enzymes responsible for writing and erasing Formyl-K108 could reveal novel druggable targets

  • Reader proteins that specifically recognize this modification represent potential intervention points

  • Understanding the genomic distribution of Formyl-K108 through ChIP-seq using validated antibodies helps prioritize therapeutic targets

  • Disease-specific alterations in this modification pathway may provide opportunities for selective therapeutic intervention

• Biomarker Development:

  • Immunohistochemical detection of Formyl-Histone H2B (K108) using validated protocols (1:50-1:800 dilution) could identify patient subgroups likely to respond to specific therapies

  • Changes in modification patterns during treatment may serve as pharmacodynamic markers

  • Combined assessment with other histone modifications could yield predictive signatures

  • Integration with genetic biomarkers may enhance precision medicine approaches

• Therapeutic Strategies:

  • Small molecule inhibitors targeting writer/eraser enzymes specific to Formyl-K108

  • Degrader technologies (PROTACs) directed against reader proteins that recognize this modification

  • Combination approaches targeting multiple histone modifications simultaneously

  • Synthetic transcription factors designed to recognize genomic regions marked by Formyl-K108

• Preclinical Validation Models:

  • Cell line panels with varying Formyl-K108 levels to screen compound efficacy

  • Patient-derived organoids to assess therapy response in more physiological contexts

  • In vivo models with genetic manipulation of the modification pathway

  • Validation using highly specific antibodies to confirm target engagement and modification changes

This research direction holds particular promise for diseases linked to histone modifications, including cancer and developmental disorders, where epigenetic dysregulation plays a causal role . The high specificity of antibodies against Formyl-Histone H2B (K108), as validated through peptide array testing against 501 different histone peptides, provides reliable tools for both target discovery and therapeutic monitoring .