Formyl-HIST1H2BC (K116) Antibody

What is Formyl-HIST1H2BC (K116) Antibody and what biological role does histone formylation play?

Formyl-HIST1H2BC (K116) Antibody is a primary antibody specifically designed to detect formylation at lysine 116 of histone H2B type 1-C/E/F/G/I in human cells . This post-translational modification occurs on histones, which are core components of nucleosomes that wrap and compact DNA into chromatin.

Histone H2B plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . Formylation represents one of the many modifications in the "histone code" that regulates DNA accessibility to cellular machinery. Histone H2B also has broad antibacterial activity and may contribute to antimicrobial barriers in colonic epithelium and amniotic fluid .

How does Formyl-HIST1H2BC (K116) Antibody differ from antibodies detecting other histone modifications?

Formyl-HIST1H2BC (K116) Antibody specifically recognizes formylation at the K116 position of HIST1H2BC, distinguishing it from antibodies targeting other modifications like 2-hydroxyisobutyrylation at K108 or formylation at K108 . The specificity of these antibodies is determined by:

• The exact position of the modification (e.g., K116 vs K108)

• The type of modification (formyl vs. 2-hydroxyisobutyryl)

• The surrounding amino acid sequence

This specificity is crucial as different modifications may have distinct functional implications in chromatin regulation. For example, while both K108 and K116 formylation occur on histone H2B, they might influence different biological processes or respond to different cellular signals .

What applications is Formyl-HIST1H2BC (K116) Antibody validated for?

The Formyl-HIST1H2BC (K116) Antibody has been validated for several experimental applications:

When using this antibody, it's important to follow recommended dilutions and protocols specific to each application to ensure optimal results. For instance, antigen retrieval methods may be necessary for certain applications like immunohistochemistry .

How can I validate the specificity of Formyl-HIST1H2BC (K116) Antibody in my experimental system?

Validating antibody specificity is crucial for accurate data interpretation. For Formyl-HIST1H2BC (K116) Antibody, consider these approaches:

• Peptide Competition Assay: Pre-incubate the antibody with formylated and non-formylated peptides containing the K116 sequence. Specific binding should be blocked only by the formylated peptide.

• Peptide Microarray Analysis: Use peptide arrays containing various histone modifications to assess cross-reactivity . The Histone Antibody Specificity Database (www.histoneantibodies.com) provides data on many histone antibodies, including their potential cross-reactivity with other modifications .

• Knockout/Knockdown Controls: Use cells where either the histone or the enzyme responsible for formylation is depleted.

• Secondary Antibody Controls: Perform parallel experiments without primary antibody to rule out non-specific binding of the secondary antibody .

• Positive Controls: Include samples known to contain high levels of the formylation mark, such as specific tissues or cell types where the modification has been well-documented .

How do neighboring post-translational modifications affect Formyl-HIST1H2BC (K116) Antibody binding?

The presence of adjacent modifications can significantly impact antibody recognition of formyl-K116. This phenomenon, known as "epitope occlusion" or "antibody epitope masking," represents a significant challenge in histone PTM research .

According to analyses from histone antibody specificity studies, antibody binding can be influenced by:

• Adjacent Modifications: Modifications at neighboring residues (e.g., K120, K112) may enhance or interfere with antibody binding to formyl-K116.

• Combinatorial Effects: Some antibodies might preferentially recognize certain combinations of modifications while failing to detect others.

• Epitope Accessibility: The three-dimensional structure of the nucleosome may affect accessibility of the formyl-K116 epitope in certain contexts.

It is recommended to validate the antibody using peptide arrays that contain various combinatorial modifications to understand these potential interactions . The Histone Antibody Specificity Database offers interactive tools to assess how neighboring modifications might influence antibody recognition .

What are the key differences between polyclonal and monoclonal Formyl-HIST1H2BC (K116) Antibodies?

The choice between polyclonal and monoclonal antibodies has significant implications for experimental outcomes:

For investigations requiring high reproducibility across experiments, recombinant monoclonal antibodies offer advantages due to their consistent production and defined specificity . Polyclonal antibodies might provide higher sensitivity but with potential variability between batches .

What protocol optimizations are recommended for Western blot using Formyl-HIST1H2BC (K116) Antibody?

For optimal Western blot results with Formyl-HIST1H2BC (K116) Antibody:

• Sample Preparation:

  • Include histone extraction buffer with deacetylase inhibitors

  • Maintain protein integrity by using protease inhibitors

  • Consider acid extraction methods specifically optimized for histones

• Blocking and Antibody Incubation:

  • For polyclonal antibodies: Optimal dilution should be determined empirically, starting with manufacturer recommendations

  • For monoclonal antibodies: Follow validated dilutions from manufacturer data

  • Use 5% BSA in TBST for blocking instead of milk (milk contains histones)

• Detection Optimization:

  • Enhanced chemiluminescence (ECL) with longer exposure times may be needed for low abundance modifications

  • Consider fluorescent secondary antibodies for more quantitative analysis

• Controls:

  • Include a non-specific IgG control from the same species as the primary antibody

  • Include a sample with known formylation status as positive control

  • Consider a competition control with formylated peptide

How can I use Formyl-HIST1H2BC (K116) Antibody in immunofluorescence and immunohistochemistry?

For immunofluorescence/immunohistochemistry applications:

• Fixation Method:

  • 4% paraformaldehyde is typically recommended for maintaining histone epitopes

  • Methanol fixation may be preferable for certain applications

• Antigen Retrieval:

  • Heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 is recommended for paraffin-embedded tissues

  • Microwave or pressure cooker methods may enhance epitope accessibility

• Antibody Dilution:

  • For IHC-P: 1/800 dilution has been validated for monoclonal antibodies

  • For IF: Start with manufacturer's recommended dilution and optimize as needed

• Visualization:

  • For brightfield microscopy: HRP-conjugated secondary antibodies with DAB or AEC

  • For fluorescence: Fluorophore-conjugated secondary antibodies matched to your microscope's filter sets

• Counterstaining:

  • Hematoxylin counterstaining helps visualize tissue architecture in IHC

  • DAPI is recommended for nuclear counterstaining in IF

How can peptide arrays be used to validate Formyl-HIST1H2BC (K116) Antibody specificity?

Peptide microarrays provide comprehensive validation of antibody specificity:

• Array Design:

  • High-density arrays containing various histone peptides with different modifications

  • Include formylated K116 peptides and related modifications

  • Incorporate peptides with neighboring modifications to test for combinatorial effects

• Experimental Procedure:

  • Incubate arrays with antibody at optimized concentration

  • Detect binding using fluorescent secondary antibodies

  • Scan arrays using appropriate imaging systems

• Data Analysis:

  • Quantify signal intensity for each peptide

  • Compare binding to target epitope versus other modifications

  • Assess influence of neighboring modifications on binding efficiency

• Resources:

  • The Histone Antibody Specificity Database provides protocols and existing data for many histone antibodies

  • Users can upload their own array results to contribute to the database using a simple CSV file format

How do I interpret Western blot results showing multiple bands with Formyl-HIST1H2BC (K116) Antibody?

Multiple bands in Western blots using Formyl-HIST1H2BC (K116) Antibody may indicate:

• Histone Variants: Different H2B variants may be detected if they share the formyl-K116 epitope

  • HIST1H2BC, HIST1H2BE, HIST1H2BF, HIST1H2BG, and HIST1H2BI share significant sequence homology

• Cross-reactivity: The antibody may recognize similar epitopes on other proteins

  • Validate using peptide competition assays or knockout controls

• Degradation Products: Histone degradation during sample preparation

  • Optimize extraction protocol and add protease inhibitors

• Other Post-translational Modifications: Similar modifications (e.g., formylation at other lysines)

  • Confirm with specific controls and peptide arrays

When interpreting results, consider comparing patterns across different cell types or treatment conditions to identify specific versus non-specific signals.

What factors might lead to false negative or weak signals when using Formyl-HIST1H2BC (K116) Antibody?

Several factors can contribute to weak or absent signals:

• Low Modification Abundance:

  • Formylation may be cell-type specific or induced under specific conditions

  • Consider enrichment strategies or more sensitive detection methods

• Epitope Masking:

  • Neighboring modifications may block antibody access to formyl-K116

  • Test sensitivity to combinatorial modifications using peptide arrays

• Fixation/Extraction Issues:

  • Inadequate fixation may lead to epitope loss

  • Improve histone extraction protocols for better yield

• Antibody-Specific Factors:

  • Storage conditions affecting antibody activity

  • Batch-to-batch variation (especially with polyclonal antibodies)

  • Incorrect dilution or incubation conditions

• Technical Considerations:

  • Insufficient antigen retrieval for fixed tissues

  • Suboptimal blocking conditions leading to high background

To troubleshoot, systematically modify each parameter while including appropriate positive controls.

How can I quantify relative levels of histone H2B formylation at K116 across different samples?

For quantitative analysis of formylation levels:

• Western Blot Quantification:

  • Use total H2B antibody as loading control

  • Calculate the ratio of formyl-K116 signal to total H2B

  • Employ image analysis software for densitometry

  • Consider using fluorescent secondary antibodies for wider linear range

• Mass Spectrometry:

  • Use heavy-isotope labeled internal standards for absolute quantification

  • Compare formylated peptide peak areas normalized to unmodified peptides

• ELISA-Based Methods:

  • Develop sandwich ELISA using capturing antibody against H2B and detection with Formyl-HIST1H2BC (K116) Antibody

  • Generate standard curves with synthetic peptides

• Immunofluorescence Quantification:

  • Measure nuclear fluorescence intensity using image analysis software

  • Normalize to total H2B or DNA content (DAPI)

  • Use consistent imaging parameters across samples

• ChIP-seq Analysis:

  • For genome-wide distribution analysis

  • Compare normalized peak heights/areas across conditions

  • Correlate with other genomic features

How does formylation at K116 differ functionally from formylation at K108 in histone H2B?

The functional differences between formylation at K116 and K108 positions remain an active area of research:

• Genomic Distribution:

  • K108 formylation may associate with different genomic regions than K116 formylation

  • Different formylation sites might correlate with distinct transcriptional states

• Protein Interactions:

  • Each modification might recruit different effector proteins

  • The structural context of K108 versus K116 within the nucleosome affects accessibility

• Regulatory Mechanisms:

  • Different enzymatic machinery may be responsible for formylation/deformylation at each site

  • Environmental triggers may differentially induce modification at specific lysines

• Cross-talk with Other Modifications:

  • The proximity of K108 and K116 to other modification sites creates different combinatorial possibilities

  • Site-specific formylation may have different relationships with other histone marks

Using site-specific antibodies for K108 and K116 formylation in parallel experiments can help elucidate these functional differences.

What is the relationship between histone H2B formylation and other epigenetic regulatory mechanisms?

Histone H2B formylation interacts with other epigenetic mechanisms:

• Integration with Histone Code:

  • Formylation may influence or be influenced by other histone modifications

  • The "histone code" represents a complex regulatory network where formylation plays a specific role

• Chromatin Structure Effects:

  • Formylation may alter nucleosome stability or positioning

  • Changes in chromatin accessibility can impact transcription factor binding

• Transcriptional Regulation:

  • Formylation patterns may correlate with gene expression states

  • Specific gene classes might be regulated by formylation-dependent mechanisms

• DNA Methylation Interaction:

  • Potential cross-talk between histone formylation and DNA methylation patterns

  • Co-occurrence or mutual exclusivity with specific DNA methylation regions

• Cellular Signaling Integration:

  • Formylation may respond to metabolic or oxidative stress signals

  • Connection to inflammation pathways given the role of formylation in bacterial defense

Understanding these relationships requires multi-omics approaches combining ChIP-seq, RNA-seq, and proteomics using Formyl-HIST1H2BC (K116) Antibody as a key reagent.

What are the current hypotheses about the enzymatic regulation of histone H2B formylation at K116?

Research into the enzymatic mechanisms of histone formylation is still evolving:

• Enzymatic Writers:

  • Unlike acetylation or methylation, specific formyltransferases for histones remain poorly characterized

  • Formylation may occur non-enzymatically under oxidative stress conditions

  • Potential role of mitochondrial enzymes in generating formyl donors

• Enzymatic Erasers:

  • Deformylases that specifically remove formyl groups from histones are being investigated

  • Some histone deacetylases may have dual specificity for formyl groups

• Regulation Mechanisms:

  • Metabolic state of the cell may influence formylation rates

  • Inflammatory processes might trigger increased histone formylation

  • Cell cycle-dependent regulation of formylation/deformylation dynamics

• Formyl Group Sources:

  • Formyl-tetrahydrofolate as potential formyl donor

  • Lipid peroxidation products as non-enzymatic formylation agents

  • Bacterial infection-induced formylation through inflammation

These hypotheses can be tested using Formyl-HIST1H2BC (K116) Antibody in combination with genetic or pharmacological manipulation of candidate enzymes.

How can Formyl-HIST1H2BC (K116) Antibody be used in single-cell analysis of histone modifications?

Emerging techniques for single-cell histone modification analysis with Formyl-HIST1H2BC (K116) Antibody:

• Single-Cell Imaging:

  • Immunofluorescence combined with high-content imaging

  • Correlative light and electron microscopy for ultrastructural localization

  • Live-cell imaging using cell-permeable antibody fragments

• Single-Cell Epigenomics:

  • CUT&Tag or CUT&RUN with Formyl-HIST1H2BC (K116) Antibody for single-cell chromatin profiling

  • Single-cell ChIP-seq adaptations using microfluidic platforms

  • Integration with single-cell RNA-seq for multi-modal analysis

• Mass Cytometry Applications:

  • Metal-conjugated Formyl-HIST1H2BC (K116) Antibody for CyTOF analysis

  • Simultaneous detection of multiple histone modifications at single-cell resolution

• Technical Considerations:

  • Antibody concentration optimization for minimizing background in single-cell applications

  • Fixation protocols that preserve both cellular morphology and epitope accessibility

  • Signal amplification methods for detecting low-abundance modifications

These approaches enable the study of cell-to-cell variation in histone formylation patterns within heterogeneous populations.

What disease associations have been identified for alterations in histone H2B formylation at K116?

While research is still emerging, several potential disease associations are being investigated:

• Cancer Biology:

  • Altered formylation patterns in certain cancer types

  • Potential prognostic value of formylation levels

  • Therapeutic implications of targeting formylation pathways

• Inflammatory Disorders:

  • Connection to intestinal inflammation given H2B's role in antimicrobial defense

  • Potential biomarker for inflammatory conditions

• Neurodegenerative Diseases:

  • Link between oxidative stress, formylation, and neurodegeneration

  • Altered chromatin regulation in neurodegenerative disorders

• Metabolic Disorders:

  • Integration of metabolic signals through histone formylation

  • Potential role in diabetes and obesity-related conditions

• Aging-Related Processes:

  • Age-dependent changes in formylation patterns

  • Connection to cellular senescence mechanisms

Formyl-HIST1H2BC (K116) Antibody serves as a critical tool for investigating these disease associations through techniques like tissue microarray analysis and biomarker validation studies.

What technical advances are improving the detection sensitivity and specificity of histone formylation using antibody-based methods?

Recent technological advances enhancing histone formylation detection:

• Antibody Engineering:

  • Development of recombinant antibodies with enhanced specificity

  • Fragment-based approaches for improved epitope access

  • Nanobodies against formylated histones for specialized applications

• Signal Amplification:

  • Tyramide signal amplification for immunohistochemistry

  • Proximity ligation assays for detecting formylation in specific contexts

  • Quantum dot-conjugated secondary antibodies for improved sensitivity

• Microfluidic Platforms:

  • Automated microfluidic immunoassays for high-throughput screening

  • Reduced sample requirements for precious specimens

• Multiplexed Detection:

  • Multiplexed immunofluorescence for simultaneous detection of multiple modifications

  • Mass spectrometry immunoassays for quantitative multiplexing

  • Sequential immunolabeling with antibody stripping protocols

• Validation Resources:

  • Expansion of databases like The Histone Antibody Specificity Database

  • Standardized reporting of antibody validation data

  • Community-driven antibody validation initiatives

These advances are collectively improving the reliability and applicability of Formyl-HIST1H2BC (K116) Antibody in diverse research contexts.