Formyl-HIST1H2AG (K118) Antibody

What is Formyl-HIST1H2AG (K118) Antibody and what specific epitope does it recognize?

Formyl-HIST1H2AG (K118) Antibody is a polyclonal antibody that specifically recognizes the formylation modification at lysine 118 of Histone H2A type 1. This antibody was developed using a peptide sequence surrounding the formyl-Lys (118) site derived from Human Histone H2A as the immunogen . The antibody targets a core component of nucleosomes, which are fundamental units of chromatin. By recognizing this specific post-translational modification, researchers can investigate how histone formylation affects chromatin structure and gene regulation. The antibody has been raised in rabbits and purified using antigen affinity methods to ensure specificity for the formylated K118 epitope .

What applications is Formyl-HIST1H2AG (K118) Antibody validated for in experimental workflows?

Formyl-HIST1H2AG (K118) Antibody has been validated for multiple experimental applications:

The antibody's application in Western blotting allows researchers to detect and quantify formylated HIST1H2AG in cell lysates, while immunofluorescence enables visualization of the spatial distribution of this modification in cellular contexts . Proper experimental design should include positive and negative controls to validate specificity, particularly when investigating novel cell types or conditions.

What is the biological significance of lysine formylation in histone biology?

Lysine formylation represents an important post-translational modification (PTM) in histone biology that has been more recently characterized compared to better-known modifications like acetylation or methylation. Histones, particularly HIST1H2AG, play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The formylation at K118 may:

• Alter nucleosome stability and chromatin compaction

• Modify the interaction between histones and DNA

• Create or disrupt binding sites for chromatin remodeling proteins

• Function as a signal in cellular stress response pathways

The strategic position of K118 within the histone structure suggests that its formylation likely impacts DNA accessibility to transcriptional machinery, potentially functioning as an epigenetic mark that regulates gene expression patterns . Unlike acetylation which neutralizes the positive charge of lysine, formylation creates a distinct chemical environment that may recruit specific reader proteins.

What are the proper storage and handling protocols for maintaining antibody efficacy?

To maintain optimal activity and specificity of Formyl-HIST1H2AG (K118) Antibody, researchers should follow these storage and handling guidelines:

• Upon receipt, store the antibody at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles, which can compromise antibody integrity

• The antibody is supplied in liquid form with a buffer containing:

  • 0.03% Proclin 300 (preservative)

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

This formulation helps maintain stability during storage. For working solutions, it is advisable to make small aliquots based on experimental needs rather than repeatedly accessing the stock solution. When handling the antibody, maintain aseptic technique to prevent microbial contamination, and avoid exposing the antibody to strong light or extreme pH conditions.

How can Formyl-HIST1H2AG (K118) Antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

While not explicitly listed among the validated applications in the provided data, adapting Formyl-HIST1H2AG (K118) Antibody for ChIP experiments requires careful optimization:

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes may need adjustment for optimal detection of formylated histones.

• Sonication parameters: Aim for chromatin fragments of 200-500bp for optimal resolution.

• Antibody concentration: Begin with 3-5μg per ChIP reaction, then titrate as needed.

• Incubation conditions: Overnight incubation at 4°C with rotation is recommended.

• Washing stringency: Include high-salt washes to minimize non-specific binding.

A recommended protocol modification includes:

Validation should include qPCR analysis of regions known to be enriched for histone H2A, followed by more comprehensive ChIP-seq analysis if broader genomic distribution is of interest.

What approaches should be used to validate the specificity of Formyl-HIST1H2AG (K118) Antibody?

Validating antibody specificity is crucial for reliable experimental results. For Formyl-HIST1H2AG (K118) Antibody, a multi-layered validation approach is recommended:

• Peptide competition assay: Pre-incubate the antibody with excess formylated K118 peptide versus unmodified peptide before immunoblotting or immunostaining. Signal reduction with the formylated peptide confirms specificity.

• Knockout/knockdown controls: Compare signals between wild-type cells and those with HIST1H2AG knockdown or knockout.

• Mass spectrometry correlation: Validate that immunoprecipitated proteins contain the expected formylated K118 modification using mass spectrometry.

• Cross-reactivity testing: Test against related histone variants and other formylated lysine sites on different histones.

• Treatment with deformylation enzymes: Compare signals before and after treatment with enzymes that remove formyl groups.

A systematic approach to validation might include:

These validation steps ensure that experimental findings reflect genuine biological phenomena rather than artifacts of non-specific antibody binding.

How does formylation at K118 interact with other histone post-translational modifications?

Histone post-translational modifications (PTMs) function within a complex, interdependent network often referred to as the "histone code." Formylation at K118 of HIST1H2AG likely participates in this code through several mechanisms:

• Competitive exclusion: Formylation at K118 physically prevents other modifications (acetylation, methylation, ubiquitination) at the same residue.

• Sequential modification: Formylation may precede or follow other modifications on nearby residues, creating specific "modification cassettes" that recruit distinct protein complexes.

• Allosteric effects: K118 formylation may induce conformational changes that alter the accessibility of other modification sites.

• Reader protein interaction: Proteins that bind formylated K118 may facilitate or inhibit the action of enzymes that modify other residues.

A potential interaction map might include:

Investigation of these interactions requires combinatorial antibody approaches, including sequential immunoprecipitation or co-staining experiments to detect co-occurrence or mutual exclusivity of modifications.

What are the optimal fixation protocols when using Formyl-HIST1H2AG (K118) Antibody for immunofluorescence?

Fixation protocols critically impact the preservation and accessibility of the formyl-K118 epitope. Based on the antibody's characteristics, the following fixation approaches are recommended:

• Paraformaldehyde (PFA) fixation:

  • 4% PFA for 10-15 minutes at room temperature

  • Short fixation times help preserve the formyl modification

  • Avoid methanol post-fixation which can extract histones

• Epitope retrieval considerations:

  • Heat-mediated antigen retrieval (citrate buffer, pH 6.0)

  • 10mM Tris, 1mM EDTA buffer (pH 9.0) can enhance signal for some cell types

  • Limited protease treatment (0.01% trypsin for 2-5 minutes) may improve epitope accessibility

• Permeabilization optimization:

  • 0.1-0.3% Triton X-100 for 5-10 minutes

  • Alternative: 0.5% saponin (gentler, may better preserve nuclear architecture)

• Blocking conditions:

  • 5% BSA or 10% normal serum from species unrelated to primary and secondary antibodies

  • Include 0.1% Tween-20 to reduce background

For immunofluorescence applications, the recommended dilution range is 1:50-1:200 . Optimal dilution should be determined experimentally for each cell type and fixation method. Extended primary antibody incubation (overnight at 4°C) often yields better signal-to-noise ratios than shorter incubations at room temperature.

What controls are essential when designing experiments with Formyl-HIST1H2AG (K118) Antibody?

Rigorous experimental design requires appropriate controls to ensure valid interpretation of results:

Additionally, validation across multiple techniques (WB, IF, ELISA) strengthens confidence in the observed patterns. For Western blotting, molecular weight verification (H2A ~14 kDa) and histone extraction controls are particularly important, as conventional protein extraction methods may not efficiently recover histones.

What factors can lead to inconsistent results, and how can these be addressed?

Several factors can contribute to variability in experimental outcomes when using Formyl-HIST1H2AG (K118) Antibody:

• Epitope accessibility issues:

  • Solution: Optimize fixation time, test multiple permeabilization methods

  • Consider mild enzymatic treatment for antigen retrieval

• Cell-cycle dependent formylation:

  • Solution: Synchronize cells or analyze subpopulations based on cell cycle markers

  • Include cell cycle phase analysis in experimental design

• Environmental influences on formylation levels:

  • Solution: Standardize culture conditions (oxygen levels, nutrient availability)

  • Document passage number and confluence when comparing results

• Technical variability in histone extraction:

  • Solution: Use specialized histone extraction kits

  • Include acid extraction methods optimized for histone recovery

• Antibody lot-to-lot variation:

  • Solution: Validate each new lot against previous results

  • Maintain reference samples for standardization

A methodical troubleshooting approach might include:

Systematic documentation of experimental conditions and regular validation of antibody performance will help maintain consistency across experiments.

How can Formyl-HIST1H2AG (K118) Antibody be integrated with other techniques for comprehensive epigenetic analysis?

Integrating multiple methodologies creates a more complete understanding of histone formylation in epigenetic regulation:

• ChIP-seq integration:

  • Combine with RNA-seq to correlate formylation with transcriptional outcomes

  • Integrate with other histone modification ChIP-seq data to build modification co-occurrence maps

• Mass spectrometry pairing:

  • Use antibody for enrichment followed by MS analysis for precise modification identification

  • Quantitative proteomics to measure formylation levels across conditions

• Live-cell imaging approaches:

  • Develop compatible protocols for fixed/live-cell transitions

  • Correlate fixed-cell antibody staining with live-cell dynamics

• Single-cell techniques:

  • Adapt protocols for single-cell Western blotting

  • Optimize for CyTOF or single-cell proteomics approaches

• Functional genomics combination:

  • Pair with CRISPR screens targeting writers/erasers/readers of histone formylation

  • Correlate with chromosome conformation capture techniques (Hi-C, 4C)

An integrated experimental approach might look like:

This multi-omic approach provides mechanistic insights beyond what any single technique can offer, creating a comprehensive view of HIST1H2AG formylation's role in chromatin biology.

What is known about the enzymes responsible for HIST1H2AG formylation and deformylation?

The enzymatic regulation of histone formylation is an emerging area of research. While specific enzymes for HIST1H2AG K118 formylation have not been fully characterized, several mechanisms may be involved:

• Potential formylation pathways:

  • Non-enzymatic formylation from formaldehyde generated during oxidative stress

  • Enzymatic transfer of formyl groups from formyl-CoA or formyl-tetrahydrofolate

  • Secondary modification resulting from formylated metabolites

• Candidate deformylation enzymes:

  • Histone deacetylases (HDACs) with potential deformylase activity

  • Dedicated histone deformylases yet to be characterized

  • Sirtuin family proteins that remove various acyl modifications

Research approaches to identify these enzymes might include:

Understanding the enzymatic regulation of this modification will provide insights into how cells regulate formylation in response to environmental cues and metabolic states.

How does HIST1H2AG formylation differ between normal and pathological states?

Histone formylation patterns may serve as biomarkers or functional contributors to disease states. While specific data on Formyl-HIST1H2AG (K118) in pathology is limited, several research directions are promising:

• Cancer biology:

  • Altered metabolism in cancer cells may affect one-carbon metabolism and formylation rates

  • Changes in formylation patterns may contribute to aberrant gene expression

  • Potential correlation with tumor aggressiveness or treatment response

• Inflammatory conditions:

  • Oxidative stress during inflammation may increase non-enzymatic formylation

  • Possible role in neutrophil extracellular trap (NET) formation

  • Contribution to altered gene expression in chronic inflammatory diseases

• Neurodegenerative disorders:

  • Connection between altered histone modifications and neurodegeneration

  • Potential accumulation of formylated histones with aging

  • Relationship to oxidative stress in Alzheimer's and Parkinson's diseases

• Metabolic disorders:

  • Link between one-carbon metabolism defects and histone formylation

  • Potential role in diabetes and obesity through epigenetic mechanisms

  • Nutritional influence on histone formylation patterns

Research strategies might include:

Using Formyl-HIST1H2AG (K118) Antibody in these contexts might reveal important disease-specific patterns and potential therapeutic targets.

What emerging technologies might enhance research using Formyl-HIST1H2AG (K118) Antibody?

As epigenetic research continues to advance, several emerging technologies may complement and enhance studies using Formyl-HIST1H2AG (K118) Antibody:

• Spatial transcriptomics/epigenomics:

  • Integration of antibody-based imaging with spatial genomics

  • Visualization of formylation patterns within tissue architecture

  • Correlation with spatially-resolved gene expression data

• Single-molecule detection:

  • Super-resolution microscopy for precise localization of formylation marks

  • Single-molecule pull-down assays for protein complex identification

  • Direct visualization of formylation dynamics in living cells

• Combinatorial modifications analysis:

  • Sequential ChIP approaches to identify co-occurrence patterns

  • Barcode-based mass cytometry for multiple modification detection

  • Proximity ligation assays to detect modification patterns in situ

• Causal testing technologies:

  • Targeted modification systems using CRISPR-based approaches

  • Optogenetic control of formylation/deformylation enzymes

  • Synthetic biology approaches to engineer specific formylation states

These technological advances will help move from correlative to causal understanding of histone formylation's role in cellular function and disease processes.

How might researchers design longitudinal studies to investigate dynamic changes in HIST1H2AG formylation?

Capturing the dynamic nature of histone formylation requires thoughtful experimental design:

• Time-course experiments:

  • Synchronized cell populations to track cell-cycle dependent changes

  • Developmental models to examine formylation during differentiation

  • Stress response time-courses to monitor acute formylation changes

• Live-cell compatible approaches:

  • Development of cell lines with fluorescent tags near modification sites

  • Adaptation of antibody fragments for live-cell imaging

  • Real-time enzymatic activity reporters for formylation/deformylation

• Pulse-chase experiments:

  • Metabolic labeling of one-carbon units to track formyl group turnover

  • Quantification of modification half-life under various conditions

  • Correlation with histone exchange rates in different genomic regions

A comprehensive longitudinal study might employ: