Formyl-HIST1H2AG (K95) Antibody

What is Formyl-HIST1H2AG (K95) Antibody and what does it recognize?

Formyl-HIST1H2AG (K95) antibody is a polyclonal antibody raised in rabbits that specifically detects endogenous levels of formylated lysine at position 95 of Histone H2A type 1 protein. The immunogen used to generate this antibody consists of a peptide sequence surrounding the formylated K95 site derived from human Histone H2A type 1 . This antibody recognizes post-translational modification on a core component of nucleosomes, which play crucial roles in chromatin structure and function.

What is the biological significance of histone H2A formylation at K95?

Histone formylation represents an important epigenetic modification that contributes to chromatin regulation. Histone H2A is a core component of nucleosomes that wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries requiring DNA as a template. Histones play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The specific formylation at K95 contributes to the complex set of post-translational modifications known as the histone code, which regulates DNA accessibility through nucleosome remodeling. While specific functions of K95 formylation are still being characterized, formylated histones may serve as important markers in DNA damage responses, cellular stress conditions, or specific transcriptional states.

How does Formyl-HIST1H2AG (K95) modification differ from other histone H2A modifications?

Formyl-HIST1H2AG (K95) represents one of several possible modifications that can occur at lysine 95 of histone H2A. Other documented modifications at this same residue include 2-hydroxyisobutyrylation , which has distinct biological functions. The formyl modification differs from other common histone modifications like acetylation, methylation, and ubiquitylation in its chemical structure and potential regulatory mechanisms. While acetylation of H2A at other lysine residues (such as H2A.X K5 by Tip60) promotes accumulation of DNA repair factors like NBS1 at double-strand breaks , formylation may have distinct roles in chromatin regulation. Unlike ubiquitylation of H2A at K13/15 by RNF168 which is involved in DNA damage response pathways , formylation represents a different class of modification with potentially specialized functions in chromatin biology.

What are the validated applications for Formyl-HIST1H2AG (K95) Antibody?

The Formyl-HIST1H2AG (K95) Antibody has been validated for several experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of formylated H2A K95 in protein samples.

• ICC (Immunocytochemistry): For cellular localization studies with recommended dilutions of 1:1-1:10.

• IF (Immunofluorescence): For visualization of the modified histone in fixed cells with recommended dilutions of 1:50-1:200 .

This polyclonal antibody specifically detects endogenous levels of Formyl-HIST1H2AG (K95) protein in human samples. Researchers should optimize dilutions based on their specific experimental conditions and cell types.

What are the recommended protocols for immunofluorescence staining with this antibody?

For optimal immunofluorescence results with Formyl-HIST1H2AG (K95) Antibody, researchers should follow this methodological approach:

• Cell preparation: Fix cells using 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 in PBS for 10 minutes.

• Blocking: Block non-specific binding using 5% normal serum (from the species of secondary antibody) in PBS with 0.1% Tween-20 for 1 hour at room temperature.

• Primary antibody incubation: Dilute the Formyl-HIST1H2AG (K95) Antibody at 1:50-1:200 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody application: After washing, apply fluorophore-conjugated secondary antibody (anti-rabbit) at manufacturer's recommended dilution for 1 hour at room temperature.

• Nuclear counterstaining: Use DAPI or other DNA stain to visualize nuclei.

• Mounting and imaging: Mount slides with anti-fade mounting medium and image using appropriate filter sets on a fluorescence or confocal microscope.

It's essential to include proper controls, including a negative control omitting primary antibody and positive control using cells known to express the target modification.

How should the antibody be stored for maximum stability and efficacy?

The Formyl-HIST1H2AG (K95) Antibody requires specific storage conditions to maintain its binding activity and specificity:

• Short-term storage: Store at +4°C for up to one week.

• Long-term storage: For extended periods, aliquot the antibody and store at -20°C or -80°C.

• Avoid freeze-thaw cycles: Each freeze-thaw cycle can cause the antibody to lose approximately half of its binding activity .

• Storage buffer: The antibody is supplied in phosphate-buffered saline (pH 7.4) containing 0.03% Proclin as preservative and 50% glycerol for stability .

Making multiple small aliquots upon initial receipt prevents repeated freeze-thaw cycles of the stock solution, thereby preserving antibody functionality for longer periods.

How can Formyl-HIST1H2AG (K95) Antibody be integrated into studies of DNA damage and repair mechanisms?

Formyl-HIST1H2AG (K95) Antibody can provide valuable insights in DNA damage and repair studies through these methodological approaches:

• Co-localization experiments: Perform double immunofluorescence staining with DNA damage markers like γH2AX to determine whether K95 formylation co-localizes with DNA double-strand breaks .

• Temporal dynamics: Conduct time-course experiments following induced DNA damage to track changes in K95 formylation patterns relative to established DNA damage response markers.

• Chromatin immunoprecipitation (ChIP): Adapt the antibody for ChIP assays to identify genomic regions where K95 formylation is enriched following DNA damage.

• Relationship to other modifications: Compare distribution patterns with other histone modifications involved in DNA repair, such as H2A ubiquitylation at K13/15 by RNF168, which is critical for 53BP1 recruitment and non-homologous end joining (NHEJ) repair .

This integrated approach would help determine whether formylation at K95 plays a regulatory role in DNA damage signaling pathways, potentially influencing repair pathway choice between homologous recombination (HR) and NHEJ.

What are the methodological considerations for studying cross-talk between formylation and other histone modifications?

To effectively study the interplay between formylation at K95 and other histone modifications, researchers should consider these methodological approaches:

• Sequential ChIP (ChIP-reChIP): Perform initial ChIP with Formyl-HIST1H2AG (K95) Antibody followed by a second round using antibodies against other modifications to identify regions with co-occurrence of multiple marks.

• Mass spectrometry analysis: Use antibody-based enrichment followed by mass spectrometry to identify peptides containing K95 formylation along with other modifications on the same histone molecule.

• Modification-specific knockdown experiments: Deplete enzymes responsible for other histone modifications and assess effects on K95 formylation patterns.

• Competitive binding assays: Similar to the competition binding assay methodology described for FPR1 ligands , develop assays to determine whether formylation at K95 affects binding of reader proteins for other modifications.

These approaches would help elucidate whether K95 formylation functions in coordination with or opposition to modifications like H2A.X S139 phosphorylation (γH2AX) or H2A K15 acetylation, which inhibits ubiquitylation and promotes homologous recombination over NHEJ .

How can Formyl-HIST1H2AG (K95) Antibody be used to study the dynamics of chromatin remodeling?

Researchers can apply Formyl-HIST1H2AG (K95) Antibody to investigate chromatin remodeling dynamics through these approaches:

• Live-cell imaging: Develop cell lines expressing fluorescently tagged chromatin remodelers and perform live imaging after immunostaining with the antibody to track temporal associations between remodelers and K95 formylation.

• Nuclear fractionation studies: Isolate chromatin fractions with different compaction states and assess the distribution of K95 formylation to determine association with euchromatin versus heterochromatin.

• Remodeler depletion experiments: Knock down or inhibit chromatin remodeling complexes like SWI/SNF or INO80, which interact with modified histones during DNA repair , and assess effects on K95 formylation patterns.

• Nucleosome positioning analysis: Combine micrococcal nuclease digestion with ChIP using the Formyl-HIST1H2AG (K95) Antibody to map the relationship between nucleosome positioning and K95 formylation.

This comprehensive approach would help determine whether K95 formylation influences or is influenced by nucleosome remodeling events, particularly during processes like DNA repair where extensive chromatin reorganization occurs.

What controls should be included when using Formyl-HIST1H2AG (K95) Antibody?

Proper experimental controls are essential when working with Formyl-HIST1H2AG (K95) Antibody:

These controls will help ensure the reliability and specificity of results obtained with the Formyl-HIST1H2AG (K95) Antibody.

How should researchers interpret variations in Formyl-HIST1H2AG (K95) signal intensity across different cell types?

When interpreting differential Formyl-HIST1H2AG (K95) staining patterns across cell types, researchers should consider:

• Baseline modification levels: Different cell types may have inherently different levels of K95 formylation based on their metabolic state and chromatin organization.

• Cell cycle dependence: Compare cells at similar cell cycle stages, as histone modifications often show cell-cycle dependent patterns.

• Normalization approach: Normalize K95 formylation signals to total H2A levels using a modification-independent H2A antibody to account for variations in histone abundance.

• Quantification method: Apply consistent quantification methods across samples, such as measuring nuclear mean fluorescence intensity or counting discrete nuclear foci.

• Biological context: Interpret variations in the context of cellular function—for example, cells with high metabolic activity or oxidative stress may show increased formylation due to elevated formyl donor availability.

What are the common technical challenges when working with Formyl-HIST1H2AG (K95) Antibody and how can they be addressed?

Researchers commonly encounter these technical challenges when working with histone modification-specific antibodies like Formyl-HIST1H2AG (K95) Antibody:

• High background signal:

  • Cause: Insufficient blocking or non-specific binding

  • Solution: Increase blocking time/concentration, optimize antibody dilution (1:50-1:200 range for IF), and ensure thorough washing

• Weak or no signal detection:

  • Cause: Epitope masking due to fixation or low abundance of modification

  • Solution: Test different fixation methods, increase antibody concentration, extend incubation time, or use signal amplification methods

• Cross-reactivity with other modifications:

  • Cause: Similar chemical structures between formylation and other lysine modifications

  • Solution: Perform peptide competition assays with specifically modified peptides to confirm antibody specificity

• Variable results between experiments:

  • Cause: Antibody degradation from improper storage or freeze-thaw cycles

  • Solution: Store antibody properly at -20°C or -80°C in small aliquots to avoid repeated freeze-thaw cycles that reduce binding activity

• Inconsistent results across cell types:

  • Cause: Differences in chromatin accessibility or epitope availability

  • Solution: Optimize fixation and permeabilization protocols for each cell type; consider antigen retrieval methods

These methodological optimizations can significantly improve experimental outcomes when working with Formyl-HIST1H2AG (K95) Antibody.

How might Formyl-HIST1H2AG (K95) relate to cellular stress responses and metabolism?

The connection between Formyl-HIST1H2AG (K95) and cellular stress/metabolism represents an emerging research area with these potential methodological approaches:

• Metabolic stress models: Expose cells to various metabolic stressors (hypoxia, nutrient deprivation, oxidative stress) and assess changes in K95 formylation patterns using the antibody.

• Correlation with formyl donors: Measure cellular formyl-donor availability (e.g., formyl-THF levels) in relation to K95 formylation patterns under different metabolic conditions.

• Integration with stress response pathways: Analyze co-localization between K95 formylation and stress-responsive transcription factors or chromatin regions using ChIP-seq or imaging approaches.

• Enzyme inhibition studies: Target enzymes potentially involved in formylation/deformylation cycles and assess impact on cellular stress resistance and recovery.

This research would help determine whether K95 formylation serves as a chromatin-based sensor of metabolic state, potentially linking changes in cellular metabolism to adaptive gene expression programs.

What is the current understanding of enzymes responsible for histone formylation and deformylation?

The enzymatic regulation of histone formylation remains an active area of investigation:

• Candidate formyltransferases: Unlike well-characterized enzymes for acetylation (HATs) or methylation (HMTs), specific formyltransferases for histones have not been definitively identified. Researchers should investigate enzymes with formyl group transfer capability from donors like formyl-THF.

• Non-enzymatic formylation: Formylation may occur through non-enzymatic processes in response to oxidative stress, similar to protein carbonylation reactions.

• Potential deformylases: Similar to histone deacetylases (HDACs) that remove acetyl groups, specific deformylases may exist to remove formyl modifications from histones.

• Experimental approaches: Candidate enzyme screening through knockdown/overexpression followed by formylation assessment using the Formyl-HIST1H2AG (K95) Antibody would help identify relevant enzymes.

Elucidating these enzymatic pathways would provide mechanistic insights into how K95 formylation is regulated and potentially enable targeted manipulation of this modification for research or therapeutic purposes.

How might Formyl-HIST1H2AG (K95) research contribute to understanding aging and age-related diseases?

Research into Formyl-HIST1H2AG (K95) may have important implications for aging studies through these research directions:

• Age-dependent formylation patterns: Compare K95 formylation levels in young versus aged tissues using the antibody to determine whether this modification changes during aging.

• Correlation with DNA damage accumulation: Analyze whether age-associated increases in DNA damage correlate with altered K95 formylation patterns, potentially linking this modification to diminished repair capacity.

• Neurodegenerative disease models: Examine K95 formylation in cellular and animal models of age-related neurodegenerative diseases, where chromatin alterations are implicated.

• Intervention studies: Test whether interventions that extend lifespan (caloric restriction, certain drugs) affect K95 formylation patterns.

This research could potentially identify K95 formylation as a novel epigenetic marker of aging processes and suggest new approaches for understanding or addressing age-related chromatin dysfunction.

What is the current state of research on histone formylation compared to other modifications?

Histone formylation represents a relatively understudied modification compared to well-established marks like acetylation, methylation, and phosphorylation. While acetylation of H2A.X at K5 by Tip60 and its role in DNA repair has been extensively characterized , specific functions of formylation at K95 require further investigation. Similarly, while the roles of H2A ubiquitylation at K13/15 by RNF168 in DNA damage signaling are well-documented , equivalent mechanistic understanding for formylation is still emerging. Tools like the Formyl-HIST1H2AG (K95) Antibody provide crucial resources for advancing this field by enabling detection and localization studies of this specific modification. As research progresses, integration of formylation into the broader histone code will help establish its functional significance in chromatin regulation and cellular processes.

What multidisciplinary approaches would advance understanding of Formyl-HIST1H2AG (K95) biology?

Advancing knowledge about Formyl-HIST1H2AG (K95) requires integration of multiple research disciplines:

• Structural biology: Determine how K95 formylation affects nucleosome structure and stability using techniques like cryo-EM or X-ray crystallography.

• Systems biology: Apply network analysis to integrate K95 formylation data with other histone modifications, transcriptional outputs, and cellular phenotypes.

• Chemical biology: Develop chemical probes that can specifically introduce or remove formyl groups at defined genomic locations.

• Computational biology: Apply machine learning approaches to predict functional consequences of K95 formylation based on its genomic distribution patterns.

• Evolutionary biology: Compare K95 conservation and formylation patterns across species to understand evolutionary significance.