Formyl-HIST1H4A (K91) Antibody

What is Histone H4 K91 formylation and its biological significance?

Histone H4 K91 formylation represents a specific post-translational modification where a formyl group is attached to the lysine residue at position 91 of the histone H4 protein. This modification plays a crucial role in chromatin organization and function. Histone H4 serves as a core component of nucleosomes, which wrap and compact DNA into chromatin structures. This compaction limits DNA accessibility to cellular machineries that require DNA as a template for processes such as transcription, replication, and repair . The formylation at K91 specifically contributes to the complex set of histone modifications collectively known as the "histone code," which regulates DNA accessibility and chromatin remodeling . The strategic positioning of K91 within the nucleosome structure suggests its involvement in nucleosome stability and potentially in regulating interactions between adjacent nucleosomes, thus influencing higher-order chromatin structure.

How does Formyl-HIST1H4A (K91) differ from other histone modifications?

Formylation at K91 of histone H4 differs from other histone modifications in several key aspects:

• Modification chemistry: Unlike acetylation or methylation, formylation involves the addition of a formyl group (CHO), creating distinct structural and functional properties.

• Position significance: K91 is located in the globular domain of histone H4 rather than in the histone tail where many other modifications occur, suggesting unique roles in nucleosome structure .

• Functional impact: While many histone tail modifications directly influence DNA-histone interactions, K91 formylation likely affects protein-protein interactions within the nucleosome core and between adjacent nucleosomes .

• Regulatory pathways: The enzymes and pathways responsible for K91 formylation appear distinct from those governing other common histone modifications, indicating separate regulatory mechanisms.

• Cross-talk potential: Peptide array analyses demonstrate that formyl-K91 exhibits specific binding profiles distinct from other modifications, suggesting unique downstream effector recruitment patterns .

What are the available types of Formyl-HIST1H4A (K91) antibodies?

Based on the current research literature, researchers can access several types of Formyl-HIST1H4A (K91) antibodies:

• Polyclonal antibodies: Examples include rabbit-derived polyclonal antibodies such as CAC15546 from Biomatik, generated against synthetic peptides containing the formyl-K91 modification .

• Monoclonal antibodies: Recombinant monoclonal antibodies such as EPR18083 (available as ab177862 and the BSA/azide-free version ab250005) provide high specificity and reproducibility for detecting this modification .

• Application-specific formulations: Different buffer formulations are available to optimize performance in specific applications, including Western blotting, immunocytochemistry, and peptide arrays .

The choice between these antibody types depends on experimental requirements, with monoclonal antibodies offering higher specificity and consistency, while polyclonal antibodies potentially provide broader epitope recognition.

What are the validated applications for Formyl-HIST1H4A (K91) antibodies?

Formyl-HIST1H4A (K91) antibodies have been validated for multiple experimental applications, each requiring specific optimization approaches:

For optimal results, researchers should validate antibody performance in their specific experimental system, as factors such as sample preparation, blocking conditions, and detection methods can significantly impact results.

How should samples be prepared for optimal Formyl-HIST1H4A (K91) detection?

Sample preparation is critical for successful detection of formyl-K91 modifications:

• Cell/tissue lysis: Use histone extraction buffers containing histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) and protease inhibitors to preserve modifications. Acidic extraction methods (e.g., 0.2M H₂SO₄ or HCl) are effective for enriching histones .

• Protein quantification: Bradford or BCA assays are suitable for histone quantification; standard curves should use the same extraction buffer as samples to ensure accuracy.

• Nucleosome preparation: For nucleosome-level studies, micrococcal nuclease digestion with carefully optimized enzyme concentration and incubation time is recommended to preserve modification integrity.

• Western blot preparation: SDS-PAGE using 15-18% gels provides optimal resolution for histone proteins. Transfer to PVDF membranes at lower voltage (30V) for longer duration (overnight) improves histone transfer efficiency .

• Fixation for microscopy: For ICC/IF applications, paraformaldehyde fixation (4%, 10 minutes) followed by permeabilization with 0.1% Triton X-100 preserves nuclear architecture while maintaining accessibility to the epitope .

Importantly, formyl modifications may be sensitive to oxidizing conditions, so samples should be processed promptly and with appropriate reducing agents when necessary.

What controls should be included when working with Formyl-HIST1H4A (K91) antibodies?

Rigorous experimental design requires appropriate controls to validate Formyl-HIST1H4A (K91) antibody results:

• Positive controls:

  • NIH/3T3 mouse embryo fibroblast cell lysates have been confirmed to express detectable levels of formyl-K91 H4

  • Recombinant histone H4 formylated at K91 (if available)

  • Synthetic peptides containing the formylated K91 epitope

• Negative controls:

  • Primary antibody omission control

  • Non-specific IgG of the same species and concentration

  • Peptide competition assay using excess unmodified and formyl-K91 peptides

  • Samples treated with deformylases (if available)

• Specificity controls:

  • Peptide array testing against multiple histone modifications to confirm specificity

  • Dot blot analysis with modified and unmodified peptides at various concentrations

  • Western blot analysis of histones with various modifications

• Validation approaches:

  • Comparison of results using multiple antibody clones targeting the same modification

  • Mass spectrometry validation of immunoprecipitated samples

  • Genetic manipulation to alter formylation levels

How can Formyl-HIST1H4A (K91) antibodies be used in chromatin immunoprecipitation (ChIP) experiments?

While not explicitly listed among the validated applications in the search results, ChIP with Formyl-HIST1H4A (K91) antibodies can be approached methodologically as follows:

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes at room temperature works for most histone modifications, but may require optimization for formyl-K91 detection. Alternative crosslinkers like EGS (ethylene glycol bis(succinimidyl succinate)) may improve results for this specific modification.

• Chromatin fragmentation: Sonication parameters should be carefully optimized to generate 200-500bp fragments without excessive heat generation that might affect modification stability. Alternatively, enzymatic fragmentation using micrococcal nuclease might preserve modification status better.

• Immunoprecipitation conditions:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Use 2-5μg antibody per ChIP reaction with 25μl of magnetic protein A/G beads

  • Include 1% BSA in IP buffer to reduce non-specific binding

  • Extend incubation time (overnight at 4°C) to improve capture efficiency

• Washing stringency: Formyl modifications may require altered washing conditions compared to other histone modifications. A titration of salt concentrations in wash buffers may be necessary to determine optimal specificity versus recovery.

• Elution and analysis: Standard elution with SDS-containing buffers at 65°C, followed by proteinase K treatment, DNA purification, and qPCR or sequencing analysis.

• Data normalization: Input normalization is essential, but additional normalization to total H4 occupancy provides more accurate interpretation of formylation changes versus histone occupancy changes.

What is the relationship between Formyl-HIST1H4A (K91) and other histone modifications?

The relationship between H4K91 formylation and other histone modifications represents an important area of investigation:

• Modification crosstalk: Peptide array analysis revealed that formyl-K91 exists within a complex modification landscape. The presence of specific modifications on adjacent residues may enhance or inhibit antibody recognition of formyl-K91, suggesting potential biological crosstalk mechanisms .

• Mutually exclusive modifications: The K91 position can undergo multiple different modifications including acetylation, methylation, and formylation. These modifications are mutually exclusive at the single-molecule level, suggesting competing regulatory pathways.

• Co-occurrence patterns: Co-immunoprecipitation followed by mass spectrometry can reveal which modifications tend to co-occur with formyl-K91 on the same nucleosome or within the same chromatin domains.

• Sequential modification: Time-course studies using synchronized cells can help determine whether formylation at K91 precedes or follows other histone modifications during processes like transcriptional activation or DNA repair.

• Functional interactions: Mutations of adjacent modification sites may affect the recognition or function of formyl-K91, providing insights into three-dimensional chromatin structure and regulation.

The specific binding profile of Formyl-HIST1H4A (K91) antibodies in peptide arrays provides valuable information about these relationships, as demonstrated in specificity testing across 501 different modified and unmodified histone peptides .

How can mass spectrometry complement antibody-based detection of Formyl-HIST1H4A (K91)?

Mass spectrometry (MS) provides powerful complementary approaches to antibody-based detection of histone H4K91 formylation:

• Confirmation of modification: MS can definitively identify the chemical nature of the modification at K91, distinguishing between formylation, acetylation, or other potential modifications with similar molecular weights.

• Quantitative analysis: MS-based approaches like Multiple Reaction Monitoring (MRM) or Parallel Reaction Monitoring (PRM) enable precise quantification of formyl-K91 levels across different biological conditions.

• Novel modification discovery: Unbiased MS approaches can reveal previously unknown modifications that co-occur with K91 formylation or identify formylation at novel sites.

• Methodology integration:

• Sample preparation considerations:

  • Propionylation of unmodified lysines improves MS detection

  • Chemical derivatization can enhance detection of formyl groups

  • Enrichment using antibodies prior to MS analysis increases sensitivity

  • Specialized fragmentation techniques (ETD/ECD) preserve labile modifications

MS analysis can validate antibody specificity while providing complementary information about modification stoichiometry and combinatorial patterns that antibody-based methods cannot easily address.

What are common issues with Formyl-HIST1H4A (K91) antibody experiments and their solutions?

Researchers frequently encounter several challenges when working with Formyl-HIST1H4A (K91) antibodies:

• High background in Western blots:

  • Solution: Use 5% BSA instead of milk for blocking, as recommended in the antibody protocols

  • Increase washing time and detergent concentration

  • Validate secondary antibody specificity and dilution

  • Pre-adsorb antibody with unmodified histone peptides

• Weak or absent signal:

  • Verify modification presence with alternative techniques

  • Optimize antibody concentration (try 1:250-1:1000 range for Western blot)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider alternative extraction methods to preserve modifications

  • Use enhanced chemiluminescence detection systems for low-abundance modifications

• Non-specific bands:

  • Perform peptide competition assays to determine specific bands

  • Use gradient gels for better separation of histones

  • Consider nuclear fractionation to enrich for histone proteins

  • Validate with knockout/knockdown controls if available

• Poor reproducibility:

  • Standardize sample processing time to minimize modification loss

  • Maintain consistent antibody lots for long-term projects

  • Include internal controls in each experiment

  • Document all processing parameters meticulously

• Cross-reactivity issues:

  • Refer to peptide array data showing antibody specificity profiles

  • Perform dot blot analysis with related modified peptides

  • Consider using monoclonal antibodies for highest specificity

  • Validate results with multiple antibody clones when available

How should Formyl-HIST1H4A (K91) antibodies be stored and handled to maintain activity?

Proper storage and handling of these antibodies is critical for maintaining their activity and specificity:

• Storage conditions:

  • Store antibodies at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles by preparing working aliquots

  • For short-term storage (1-2 weeks), 4°C is acceptable

  • Protect from light, especially fluorophore-conjugated antibodies

• Working solution preparation:

  • Use high-quality, nuclease-free, sterile buffers

  • Include appropriate preservatives (0.02% sodium azide) for working solutions

  • Prepare fresh dilutions for each experiment when possible

  • Use standard antibody dilution buffers with 1-5% BSA

• Stability considerations:

  • Monitor expiration dates provided by manufacturers

  • Document lot numbers and perform lot-to-lot validation

  • Test activity periodically on positive control samples

  • Consider adding stabilizing proteins (e.g., BSA) to diluted antibodies

• Shipping and temporary storage:

  • Follow manufacturer recommendations for reconstitution

  • Allow refrigerated antibodies to equilibrate to room temperature before opening

  • Centrifuge vials briefly before opening to collect solution

  • Return to recommended storage conditions promptly after use

• Quality control measures:

  • Periodically test antibody performance against reference standards

  • Consider including antibody validation controls in routine experiments

  • Document antibody performance in laboratory notebooks

  • Create internal reference samples for long-term consistency assessment

How can quantitative analysis of Formyl-HIST1H4A (K91) be performed reliably?

Reliable quantification of H4K91 formylation levels requires careful methodological considerations:

• Western blot quantification:

  • Use internal loading controls (total H4 or other stable proteins)

  • Perform technical replicates (minimum 3) and biological replicates (minimum 3)

  • Ensure signal is within linear range of detection

  • Use digital imaging systems rather than film for better quantitative accuracy

  • Normalize formyl-K91 signal to total H4 signal from the same membrane or a parallel membrane

• ELISA-based quantification:

  • Generate standard curves using synthetic formylated peptides

  • Include spike-in controls to assess recovery efficiency

  • Validate antibody specificity in the ELISA format

  • Consider competitive ELISA formats for increased specificity

• Immunofluorescence quantification:

  • Use consistent exposure settings between samples

  • Normalize to DAPI or total H4 staining

  • Employ automated image analysis algorithms

  • Account for cell cycle variations when analyzing nuclear signals

  • Use Z-stack acquisitions for accurate nuclear signal integration

• Statistical analysis recommendations:

  • Apply appropriate statistical tests based on data distribution

  • Use non-parametric tests if normality cannot be confirmed

  • Account for multiple testing corrections in genome-wide studies

  • Report effect sizes along with p-values

  • Clearly document all normalization procedures

• Integrated multi-method approach:

  • Validate quantitative findings using complementary techniques

  • Consider absolute quantification using MS-based approaches

  • Develop consistent protocols for inter-laboratory comparison

  • Document all parameters that might affect quantification

What cellular processes are associated with changes in Formyl-HIST1H4A (K91)?

The cellular contexts in which H4K91 formylation changes have been observed provide insights into its biological functions:

• Transcriptional regulation: The position of K91 within the nucleosome core can potentially affect DNA wrapping and accessibility to transcription factors. Changes in formylation levels correlate with transcriptional activity in specific genomic regions .

• DNA damage response: Formylation may be induced by oxidative damage and serve as a signal for repair machinery recruitment. The timing of formylation changes during DNA damage response suggests both signaling and structural roles.

• Chromatin compaction states: The strategic location of K91 at the interface between histones suggests a role in nucleosome stability and higher-order chromatin structure, potentially influencing chromatin compaction and decompaction during various cellular processes.

• Cell cycle progression: Formylation levels at K91 may vary throughout the cell cycle, particularly during S-phase when chromatin is being replicated and reassembled.

• Cellular metabolism: Formylation requires formyl donors, potentially linking this modification to cellular metabolic states and mitochondrial function. Changes in cellular metabolism could affect the availability of formyl groups and therefore the levels of H4K91 formylation.

Understanding these contexts requires integrated experiments combining H4K91 formylation detection with functional assays for specific cellular processes.

How does Formyl-HIST1H4A (K91) compare between different species and cell types?

Comparative analysis of H4K91 formylation across species and cell types reveals important evolutionary and developmental patterns:

• Species conservation: The antibodies have been validated for reactivity with human and mouse samples , suggesting conservation of this modification. The high conservation of histone H4 sequence across species suggests the modification may be present in other organisms as well.

• Cell type variation: Different cell types show varying baseline levels of H4K91 formylation, potentially reflecting their different chromatin states and metabolic profiles:

  • NIH/3T3 mouse embryo fibroblasts show detectable levels by Western blot

  • Stem cells may exhibit distinct patterns associated with pluripotency

  • Terminally differentiated cells often display more stable modification patterns

• Developmental dynamics: Changes in formylation patterns during development and differentiation may reflect changing chromatin requirements:

  • Early embryonic cells undergo extensive chromatin remodeling

  • Lineage specification involves establishment of cell-type-specific modification patterns

  • Aging cells show altered histone modification landscapes

• Pathological variations: Aberrant formylation patterns may be associated with disease states:

  • Cancer cells often display altered histone modification profiles

  • Neurodegenerative conditions involve changes in chromatin regulation

  • Inflammatory conditions may affect formylation through metabolic changes

• Experimental approaches for comparative studies:

  • Cross-species Western blot analysis with standardized loading

  • Immunohistochemistry of tissue panels with quantitative image analysis

  • Mass spectrometry-based comparative histone profiling

  • Integrated genomics approaches (ChIP-seq, RNA-seq) across cell types

What are emerging research directions for Formyl-HIST1H4A (K91) studies?

Several promising research frontiers are emerging in the study of H4K91 formylation:

• Enzymology of formylation/deformylation: Identification and characterization of the enzymes responsible for adding and removing formyl groups at K91 would provide crucial mechanistic insights and potential therapeutic targets.

• Reader proteins: Discovery of proteins that specifically recognize and bind to formylated K91 would illuminate downstream signaling pathways and functional consequences of this modification.

• Single-cell analysis: Development of methods to detect H4K91 formylation at the single-cell level would reveal cell-to-cell variation and heterogeneity within populations.

• Genome-wide mapping: ChIP-seq studies with Formyl-HIST1H4A (K91) antibodies would reveal the genomic distribution of this modification and its correlation with gene expression and chromatin states.

• Crosstalk mechanisms: Systematic investigation of how H4K91 formylation interacts with other histone modifications would uncover regulatory networks within the histone code.

• Metabolic connections: Exploring how cellular metabolism influences H4K91 formylation through changes in formyl donor availability would connect chromatin regulation with broader cellular physiology.

• Technological innovations:

  • Development of formylation-specific biosensors for live-cell imaging

  • CRISPR-based approaches to manipulate formylation at specific genomic loci

  • Antibody engineering to improve specificity and sensitivity

  • Novel chemical biology tools to detect and manipulate formylation

• Therapeutic implications: Understanding the role of H4K91 formylation in disease contexts could lead to novel diagnostic markers or therapeutic strategies targeting chromatin regulation.