Formyl-HIST1H3A (K122) Antibody

What is Formyl-HIST1H3A (K122) Antibody and what does it detect?

Formyl-HIST1H3A (K122) Antibody is a specialized immunological reagent that specifically recognizes and binds to histone H3.1 (HIST1H3A) when it contains a formyl group modification at lysine 122. This antibody enables researchers to study this specific post-translational modification of histone proteins . The antibody targets a core component of nucleosomes, which are fundamental units of chromatin structure that wrap and compact DNA, thereby regulating DNA accessibility to cellular machinery involved in transcription, replication, and repair .

The target protein (Histone H3.1) is identified by UniProt ID P68431 and has multiple synonyms including Histone H3/a, Histone H3/b, Histone H3/c, and others . The antibody is typically raised against synthetic peptides derived from human HIST1H3A containing the formylated lysine at position 122, such as the sequence IMP-(Fo)K-DI .

What are the common applications for Formyl-HIST1H3A (K122) Antibody?

Formyl-HIST1H3A (K122) Antibody has been validated for several key research applications:

These applications enable researchers to investigate the presence, abundance, and localization of H3K122 formylation in various experimental contexts . The antibody has demonstrated reactivity with human samples, and some versions may cross-react with mouse and rat samples due to the high conservation of histone sequences across species .

What is the biological significance of histone H3.1 formylation at lysine 122?

Lysine 122 is located in a critically important region of histone H3.1, positioned at the nucleosome dyad axis where DNA makes contact with the histone octamer. Formylation at this site has significant implications for chromatin structure and function:

• Chromatin Structure Modulation: Formylation at K122 can directly affect nucleosome stability by altering the electrostatic interactions between histones and DNA .

• Transcriptional Regulation: As part of the "histone code," this modification plays a role in regulating gene expression by influencing the accessibility of DNA to transcription factors and other regulatory proteins .

• Cancer and Disease Associations: Altered patterns of histone formylation have been implicated in various pathological conditions, particularly cancer, where they may contribute to dysregulated gene expression .

Understanding the presence and dynamics of this modification provides insights into fundamental epigenetic mechanisms that control cellular processes and may be dysregulated in disease states .

How should I store and handle Formyl-HIST1H3A (K122) Antibody to maintain its activity?

Proper storage and handling are crucial for maintaining antibody performance:

• Storage Temperature: Store at -20°C to -70°C for long-term preservation .

• Avoid Freeze-Thaw Cycles: Minimize repeated freezing and thawing which can degrade antibody quality .

• Working Aliquots: Upon receipt, divide into small aliquots to prevent repeated freeze-thaw cycles.

• Buffer Conditions: Formyl-HIST1H3A (K122) Antibodies are typically provided in buffers containing PBS, pH 7.4, with 150mM NaCl and often 50% glycerol as a cryoprotectant .

• Handling: Work in clean environments to prevent contamination, and use proper aseptic techniques when accessing antibody solutions.

Following these storage and handling guidelines will help ensure consistent experimental results and extend the useful life of the antibody preparation.

How can I validate the specificity of Formyl-HIST1H3A (K122) Antibody in my experimental system?

Comprehensive validation of antibody specificity is essential for generating reliable research data. The following methodological approach is recommended:

• Peptide Competition Assay: Pre-incubate the antibody with increasing concentrations of the immunizing peptide (formylated K122 peptide) before application in your detection method. Signal reduction confirms specificity for the formylated epitope .

• Cross-Reactivity Assessment: Test against both formylated and unmodified peptides, as well as peptides with other modifications at K122 (acetylation, methylation) to ensure the antibody discriminates between different modifications .

• Knockout/Knockdown Controls: When possible, use genetic models where the histone variant is depleted or where enzymes responsible for formylation are knocked out.

• Mass Spectrometry Correlation: Validate antibody-based detection with mass spectrometry analysis of histone modifications to confirm the presence of formylation at K122.

• Multiple Antibody Comparison: When available, compare results using different antibody clones (such as comparing monoclonal 32C1 with other antibodies targeting the same modification) .

A comprehensive validation plan incorporates multiple complementary approaches to ensure antibody specificity before proceeding with extensive experimental work.

What are the optimal protocols for immunohistochemistry using Formyl-HIST1H3A (K122) Antibody?

For successful immunohistochemistry with Formyl-HIST1H3A (K122) Antibody, consider the following protocol optimizations:

• Tissue Preparation:

  • Fix tissues promptly (preferably in 10% neutral buffered formalin)

  • Limit fixation time to prevent epitope masking

  • Use positively charged slides for better tissue adherence

• Antigen Retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0-9.0)

  • Optimize retrieval time (typically 15-20 minutes at 95-100°C)

• Blocking and Permeabilization:

  • Block with 5-10% normal serum from the species of the secondary antibody

  • Include 0.1-0.3% Triton X-100 for nuclear permeabilization

• Antibody Incubation:

  • Dilute antibody 1:50-1:200 in blocking buffer

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Include positive controls (tissues known to express formylated H3K122)

  • Include negative controls (primary antibody omission and competitive blocking)

• Detection System:

  • Use high-sensitivity detection systems (e.g., polymer-based)

  • Optimize counterstaining to visualize nuclear morphology without obscuring specific signals

• Signal Evaluation:

  • Assess nuclear localization of staining

  • Document staining intensity and distribution patterns

  • Consider digital image analysis for quantification

These methodological details will help researchers achieve optimal staining results when using Formyl-HIST1H3A (K122) Antibody for IHC applications.

How does histone H3K122 formylation relate to other histone modifications in the epigenetic landscape?

Histone H3K122 formylation exists within a complex network of histone modifications that collectively regulate chromatin structure and function:

• Spatial Context: K122 is located at the nucleosome dyad axis, a region where DNA makes direct contact with the histone octamer. Modifications at this site can directly influence nucleosome stability and DNA accessibility, distinct from modifications on histone tails .

• Functional Relationship with Other Modifications:

  • Acetylation at K122: While K122 acetylation promotes transcriptional activation by destabilizing nucleosomes, formylation may have distinct effects

  • Crosstalk with Tail Modifications: H3K122 formylation likely functions in concert with modifications on histone tails (e.g., H3K4me3, H3K27ac) to fine-tune gene expression

  • Competitive Modifications: Formylation, acetylation, and other modifications at K122 are mutually exclusive, suggesting regulatory competition

• Regulatory Enzymes:

  • Unlike well-characterized "writers" and "erasers" for acetylation and methylation, the enzymatic machinery responsible for formylation/deformylation is less well characterized

  • Potential links to metabolic state and oxidative stress have been proposed

• Temporal Dynamics:

  • Evidence suggests that formylation may represent a more stable modification compared to the more dynamic acetylation, potentially serving as a longer-term epigenetic mark

Understanding how H3K122 formylation integrates with other histone modifications provides crucial context for interpreting experimental results and designing studies to elucidate specific functional consequences.

What research areas are currently exploring H3K122 formylation using this antibody?

Formyl-HIST1H3A (K122) Antibody has been utilized in several cutting-edge research areas:

• Cancer Biology: Investigations into altered histone formylation patterns in various cancer types, particularly breast cancer, where this modification may contribute to oncogenic gene expression programs .

• Stem Cell Research: Studies examining the role of histone formylation in maintaining pluripotency and during cellular differentiation processes .

• Chromatin Architecture: Research exploring how formylation at the nucleosome dyad affects higher-order chromatin structure and nuclear organization.

• Transcriptional Regulation: Investigations into the impact of H3K122 formylation on RNA polymerase accessibility and activity at specific genomic loci.

• Metabolic Regulation of Epigenetics: Studies examining how cellular metabolic state influences histone formylation through changes in formyl-donor availability.

• DNA Damage Response: Research into potential roles of H3K122 formylation in DNA repair processes and genomic stability maintenance.

These diverse research directions highlight the importance of having reliable antibodies against formylated H3K122 to advance understanding of this epigenetic modification's biological significance.

What are effective troubleshooting strategies for experiments using Formyl-HIST1H3A (K122) Antibody?

When encountering challenges with Formyl-HIST1H3A (K122) Antibody experiments, consider the following methodological troubleshooting approaches:

Implementing these targeted troubleshooting strategies can help researchers overcome technical challenges and generate reliable data when working with Formyl-HIST1H3A (K122) Antibody.

How should I design ChIP-seq experiments using Formyl-HIST1H3A (K122) Antibody?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with Formyl-HIST1H3A (K122) Antibody requires careful experimental design:

• Starting Material Optimization:

  • Use 1-5 million cells per immunoprecipitation

  • Ensure high cell viability before fixation

  • Consider cell type-specific optimization (cancer vs. normal cells)

• Crosslinking and Chromatin Preparation:

  • Optimize formaldehyde concentration (typically 1%) and fixation time (8-10 minutes)

  • Ensure efficient sonication to generate 200-500 bp fragments

  • Verify fragment size distribution by agarose gel electrophoresis

• Immunoprecipitation Protocol:

  • Pre-clear chromatin with protein A/G beads

  • Determine optimal antibody amount (typically 2-5 μg per IP)

  • Include appropriate controls:

    • IgG negative control

    • Input DNA control

    • Total H3 antibody for normalization

    • Positive control targeting known abundant modifications (e.g., H3K4me3)

• Library Preparation and Sequencing Considerations:

  • Use spike-in normalization standards for quantitative comparisons

  • Sequence to adequate depth (minimum 20-30 million reads)

  • Include biological replicates (minimum 2-3)

• Data Analysis Approach:

  • Normalize to input and IgG controls

  • Consider integrative analysis with RNA-seq and other histone marks

  • Analyze genomic distribution relative to transcriptional elements

This methodological framework provides researchers with a starting point for designing rigorous ChIP-seq experiments to investigate the genomic distribution of H3K122 formylation.

What considerations are important when comparing different histone H3 modification antibodies in multiplex experiments?

When designing multiplex experiments to examine relationships between H3K122 formylation and other histone modifications, consider these methodological aspects:

• Antibody Compatibility Assessment:

  • Verify host species compatibility for multi-color immunofluorescence

  • Test for potential cross-reactivity between antibodies

  • Validate each antibody individually before combining

• Sequential ChIP Design (Re-ChIP):

  • Consider epitope accessibility in sequential immunoprecipitations

  • Optimize elution conditions between IPs to preserve epitopes

  • Verify recovery efficiency at each step

• Multiplexed Detection Systems:

  • For imaging applications:

    • Select fluorophores with minimal spectral overlap

    • Include appropriate single-stain controls

    • Perform sequential staining when using same-species antibodies

  • For flow cytometry:

    • Establish compensation controls

    • Validate signal specificity with blocking peptides

    • Consider fixation/permeabilization effects on epitope detection

• Control for Biological Variables:

  • Account for cell cycle effects on histone modification patterns

  • Consider treatment timing when studying dynamic modifications

  • Include synchronization protocols when appropriate

• Data Normalization Strategies:

  • Normalize modification-specific signals to total histone H3

  • Use spike-in controls for quantitative comparisons

  • Implement batch correction for experiments performed across multiple days

These methodological considerations enable researchers to generate reliable comparative data on H3K122 formylation in relation to other histone modifications.

How can I quantitatively assess H3K122 formylation levels in different experimental conditions?

For quantitative analysis of H3K122 formylation across experimental conditions, implement these methodological approaches:

• Western Blot Quantification:

  • Extract histones using acid extraction protocols

  • Separate using SDS-PAGE with 15-18% gels for optimal histone resolution

  • Transfer to PVDF membranes (recommended over nitrocellulose for histones)

  • Probe with Formyl-HIST1H3A (K122) Antibody at 1:500-1:2000 dilution

  • Normalize to total H3 levels using a modification-insensitive H3 antibody

  • Use fluorescent secondary antibodies for wider linear detection range

  • Include standard curves with recombinant histones when possible

• ELISA-Based Approaches:

  • Develop sandwich ELISA with capture antibody against H3 and detection with Formyl-HIST1H3A (K122) Antibody

  • Generate standard curves using synthetic formylated peptides

  • Normalize to total histone content

  • Consider commercial histone modification ELISA kits with compatible antibodies

• Mass Spectrometry Analysis:

  • Perform histone propionylation to improve peptide properties for LC-MS/MS

  • Use antibody-based enrichment before MS analysis for low-abundance modifications

  • Implement parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) for targeted quantification

  • Calculate modification stoichiometry by comparing modified to unmodified peptides

• Imaging-Based Quantification:

  • Standardize image acquisition parameters across all samples

  • Implement nuclear segmentation for single-cell analysis

  • Normalize formylation signal to DAPI or total H3 staining

  • Use automated image analysis pipelines to reduce bias

These quantitative methodologies provide researchers with reliable approaches to measure changes in H3K122 formylation levels under different experimental conditions.

What is known about the relationship between histone H3K122 formylation and chromatin remodeling?

The relationship between H3K122 formylation and chromatin remodeling represents an emerging area of research:

• Structural Implications:

  • H3K122 is located at the nucleosome dyad axis where DNA makes crucial contacts with the histone octamer

  • Formylation neutralizes the positive charge of lysine, potentially weakening histone-DNA interactions

  • This modification may create a more permissive chromatin structure for remodeling enzyme access

• Interplay with Chromatin Remodeling Complexes:

  • Emerging evidence suggests that H3K122 formylation may influence the recruitment or activity of ATP-dependent chromatin remodeling complexes

  • The precise mechanisms of this interaction remain to be fully elucidated through biochemical and structural studies

• Functional Consequences:

  • Altered nucleosome positioning and stability

  • Modified higher-order chromatin structure

  • Potentially increased accessibility for transcription factors and the transcriptional machinery

• Methodological Approaches to Study This Relationship:

  • In vitro nucleosome reconstitution with formylated H3K122

  • Nucleosome remodeling assays comparing unmodified vs. formylated templates

  • Genome-wide nucleosome mapping in contexts with altered H3K122 formylation

These insights provide a foundation for designing experiments to further elucidate the functional significance of H3K122 formylation in chromatin dynamics and gene regulation.

How can I design experiments to investigate the writers and erasers of H3K122 formylation?

Unlike many histone modifications with well-characterized enzymatic machinery, the specific writers and erasers of H3K122 formylation remain less defined. Consider these experimental approaches:

• Candidate Enzyme Screening:

  • Generate a list of potential formyltransferases based on structural similarities to known histone-modifying enzymes

  • Perform siRNA/CRISPR knockdown screens of candidate enzymes

  • Quantify changes in H3K122 formylation levels using the antibody in Western blot or immunofluorescence assays

• Metabolic Manipulation Experiments:

  • Modulate cellular one-carbon metabolism (potential source of formyl groups)

  • Trace experiments with isotope-labeled metabolic precursors

  • Monitor H3K122 formylation levels under various metabolic states

• Mass Spectrometry-Based Enzyme Discovery:

  • Implement affinity purification using modified and unmodified H3K122 peptides

  • Identify differentially bound proteins by mass spectrometry

  • Validate candidates through biochemical assays

• In Vitro Enzymatic Assays:

  • Develop assays using recombinant histone H3 as substrate

  • Test candidate enzymes for formyltransferase or deformylase activity

  • Confirm site specificity using mutated histones (K122A, K122R)

• Temporal Dynamics Analysis:

  • Establish cell systems with inducible expression of tagged histones

  • Track modification appearance and disappearance kinetics

  • Correlate with cellular conditions or stimuli

These systematic approaches provide a framework for identifying and characterizing the enzymatic machinery responsible for regulating H3K122 formylation.

What are the latest findings on the role of H3K122 formylation in disease pathogenesis?

Research into the role of H3K122 formylation in disease contexts is still developing, with several emerging areas of investigation:

• Cancer Associations:

  • Altered H3K122 formylation patterns have been observed in various cancer types, particularly breast cancer

  • Studies suggest potential associations with oncogenic gene expression programs

  • Initial evidence links formylation changes to tumor progression and metastatic potential

• Inflammatory Conditions:

  • Emerging evidence suggests connections between oxidative stress, inflammation, and histone formylation

  • Changes in one-carbon metabolism during inflammation may influence formylation levels

  • Potential role in inflammatory gene regulation programs

• Neurodegenerative Disorders:

  • Preliminary studies exploring connections between altered histone formylation and neurodegenerative processes

  • Potential links to mitochondrial dysfunction and oxidative stress in neuronal cells

• Metabolic Disorders:

  • Initial investigations into how metabolic dysregulation affects histone formylation patterns

  • Possible connections to insulin resistance and diabetes pathogenesis

• Research Methodologies in Disease Contexts:

  • Patient-derived tissue analysis using Formyl-HIST1H3A (K122) Antibody

  • Integration of genomic and epigenomic data from disease cohorts

  • Disease model systems to study functional consequences of altered formylation

This evolving understanding provides opportunities for researchers to explore H3K122 formylation as both a biomarker and potential therapeutic target in various disease contexts.

What are appropriate positive and negative controls when using Formyl-HIST1H3A (K122) Antibody?

Implementing robust controls is essential for reliable interpretation of results with Formyl-HIST1H3A (K122) Antibody:

Positive Controls:

• Cell Line Standards:

  • Cell lines with known high levels of H3K122 formylation (based on literature)

  • Cells treated with inducers of histone formylation (e.g., oxidative stress agents)

• Recombinant/Synthetic Standards:

  • Formylated H3K122 peptides (such as the immunogen peptide)

  • Formylated recombinant histone H3

  • Control protein microarrays containing formylated and unmodified peptides

• Tissue Standards:

  • Tissues with documented H3K122 formylation (based on literature)

  • Serial sections of positive control tissues for IHC optimization

Negative Controls:

• Antibody Controls:

  • Primary antibody omission

  • Isotype control antibody (matching host species and isotype)

  • Pre-immune serum (for polyclonal antibodies)

• Peptide Competition:

  • Antibody pre-incubated with excess formylated H3K122 peptide

  • Comparison with unformylated peptide pre-incubation

• Genetic Controls:

  • Cells with H3.1 knockdown/knockout (when available)

  • K122 mutant constructs (K122A, K122R) expressed in cells

• Technical Controls:

  • Secondary antibody-only controls

  • Blocking peptide controls

  • Cross-reactivity assessment with other modified peptides

Implementing these control strategies provides crucial validation of signal specificity and technical reliability in experiments using Formyl-HIST1H3A (K122) Antibody.

How do different sample preparation methods affect the detection of H3K122 formylation?

Sample preparation significantly impacts the detection of histone modifications, including H3K122 formylation:

• Fixation Effects:

  • Formaldehyde Fixation: Common for IHC and IF, but may mask epitopes; requires optimization of fixation time (typically 10-15 minutes)

  • Methanol Fixation: Preserves some histone epitopes better than formaldehyde but can cause protein precipitation

  • Fresh-Frozen Samples: May preserve native epitopes but compromise morphology

  • FFPE Tissues: Require robust antigen retrieval methods to expose the H3K122 formylation epitope

• Antigen Retrieval Methods:

  • Heat-Induced Epitope Retrieval (HIER): Most effective for histone modifications in FFPE tissues

  • Citrate Buffer (pH 6.0): Standard starting point for histone modifications

  • EDTA Buffer (pH 8-9): Alternative when citrate buffer yields suboptimal results

  • Enzymatic Retrieval: Generally less effective for histone modifications

• Histone Extraction Protocols:

  • Acid Extraction: Gold standard for histone preparation in biochemical analyses

  • Triton Extraction: Preserves nuclear architecture for imaging applications

  • Direct Lysis: May result in lower sensitivity due to dilution of histones in whole cell lysates

• Storage Considerations:

  • Freeze-Thaw Effects: Multiple cycles can degrade histone modifications

  • Long-term Storage: Stability of formylation during prolonged storage requires investigation

  • Stability in Paraffin Blocks: Epitope may deteriorate in very old archived samples

Optimizing sample preparation protocols is essential for reliable detection of H3K122 formylation and should be carefully considered in experimental design.