Formyl-HIST1H4A (K12) Antibody

What is the Formyl-HIST1H4A (K12) antibody and what does it detect?

The Formyl-HIST1H4A (K12) antibody is a polyclonal antibody that specifically recognizes formylated lysine at position 12 of human histone H4 (HIST1H4A). This antibody detects a specific post-translational modification (PTM) that occurs on histone H4, which is part of the nucleosome core particle in chromatin structure. The antibody is typically raised in rabbits using a peptide sequence around the site of Formyl-Lys (12) derived from Human Histone H4 as the immunogen . This specific antibody provides researchers with a tool to investigate formylation at K12, which represents an important epigenetic mark that may influence chromatin organization and gene expression.

What are the common applications for Formyl-HIST1H4A (K12) antibody?

The Formyl-HIST1H4A (K12) polyclonal antibody has been validated for several standard laboratory applications in epigenetics and molecular biology research:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of formylated H4K12 in cell or tissue lysates

• Western Blotting (WB): For detecting the presence and relative abundance of formylated H4K12 in protein samples

• Immunocytochemistry (ICC): For visualizing the cellular distribution of formylated H4K12 in fixed cells

• Immunofluorescence (IF): For fluorescent visualization of formylated H4K12 localization in cells or tissues

When designing experiments, researchers should consider that the antibody specifically recognizes human (Homo sapiens) histone H4 with formylation at the K12 position, which may limit cross-reactivity with other species or modifications.

What is the biological significance of histone H4 K12 formylation compared to other modifications?

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, with each modification potentially serving distinct biological functions . While acetylation at K12 has been extensively studied, formylation represents a distinct modification with potentially different roles. Newly synthesized histone H4 is typically diacetylated at K5/K12 in diverse organisms, a pattern historically associated with histone deposition during DNA replication .

How should researchers optimize experimental conditions when using Formyl-HIST1H4A (K12) antibody for chromatin immunoprecipitation (ChIP) assays?

While the product information indicates the antibody has been validated for ELISA, WB, ICC, and IF applications , researchers interested in using it for ChIP should first validate its specificity in this context. For optimal ChIP protocol development:

• Crosslinking optimization: Start with standard formaldehyde crosslinking (1% for 10 minutes at room temperature), but consider titrating concentration and time for histone formylation studies.

• Sonication parameters: Aim for chromatin fragments of 200-500bp. This typically requires optimization with your specific sonicator settings.

• Antibody concentration: Begin with 2-5μg of antibody per ChIP reaction and adjust based on preliminary results. Include appropriate controls:

  • Input chromatin control (pre-immunoprecipitation)

  • Negative control using non-specific IgG

  • Positive control using an antibody against a known abundant histone mark

• Washing stringency: Balance between minimizing background and preserving specific interactions. Consider a titration of salt concentrations in wash buffers.

• Validation: Confirm specificity using:

  • Western blot of input and immunoprecipitated material

  • qPCR of regions known to be enriched or depleted for this mark

  • Peptide competition assays with formylated and unmodified peptides

The success of ChIP experiments with this antibody will depend on the abundance of the formyl-K12 mark and potential epitope masking within chromatin complexes.

What are the key considerations when designing experiments to investigate the relationship between histone H4 K12 formylation and nucleosome assembly?

Research on the analogous acetylation at K12 has shown that this modification alone is not required for nucleosome assembly in yeast . When designing experiments to investigate whether formylation at K12 influences nucleosome assembly, consider these methodological approaches:

• In vivo superhelicity assays: Measure plasmid superhelical density in whole cells as an indicator of nucleosome formation, similar to methods used to demonstrate that K5/K12 mutations do not prevent nucleosome assembly in yeast . This approach requires:

  • Construction of cellular systems with mutations at the K12 site that prevent formylation

  • Extraction of plasmid DNA under conditions that preserve supercoiling

  • Analysis by agarose gel electrophoresis with chloroquine to resolve topoisomers

• In vitro nucleosome assembly assays: Using purified components to assess whether formylation at K12 influences assembly kinetics or structural outcomes:

  • Compare assembly efficiency using histone H4 with and without formylation at K12

  • Analyze by native gel electrophoresis, MNase digestion patterns, and salt-dependent stability assays

• Combined mutation approaches: Based on findings that K5, K8, and K12 function redundantly in nucleosome assembly , design experiments that systematically examine combinations of modifications:

  • Create systems with mutations preventing formylation at multiple sites

  • Test for synthetic phenotypes that appear only when multiple sites are mutated

  • Consider the influence of the histone H3 N-terminus, which has functions redundant with H4 in histone deposition

How can researchers differentiate between formylation and acetylation at H4K12 in their experimental analyses?

Distinguishing between formylation and acetylation at the same residue presents significant technical challenges. A methodological approach should include:

• Antibody specificity validation:

  • Perform peptide competition assays using synthetic peptides containing either formylated or acetylated K12

  • Conduct Western blot analysis comparing known formylated and acetylated samples

  • Consider dot blot analysis with modified and unmodified peptides at varying concentrations

• Mass spectrometry approaches:

  • Utilize high-resolution mass spectrometry to distinguish between formylation (mass shift of +28 Da) and acetylation (mass shift of +42 Da)

  • Implement targeted MS/MS analysis focusing specifically on the K12-containing peptide

  • Consider chemical derivatization strategies that can selectively react with either formyl or acetyl groups

• Sequential immunoprecipitation:

  • First immunoprecipitate with anti-acetyl-K12 antibody

  • Then immunoprecipitate the unbound fraction with anti-formyl-K12 antibody

  • Analyze both fractions to assess relative abundance and distribution

• Enzymatic manipulation:

  • Treat samples with histone deacetylases (HDACs) that remove acetyl but not formyl groups

  • Compare antibody reactivity before and after HDAC treatment

How should researchers interpret conflicting results between formylation detection at H4K12 and functional outcomes in nucleosome assembly?

When facing contradictory results between the detection of formylation at H4K12 and functional outcomes in nucleosome assembly experiments, consider these analytical approaches:

• Assess modification abundance: The functional impact of a modification often depends on its stoichiometry. Quantitative approaches like mass spectrometry can determine what percentage of H4K12 is actually formylated in your experimental system.

• Consider context dependency: The research on H4K5/K12 acetylation demonstrates that these modifications function redundantly with K8 in nucleosome assembly . Similarly, the functional significance of H4K12 formylation might depend on the modification state of neighboring residues. Analyze:

  • Co-occurrence patterns with other modifications

  • Cell cycle phase-specific effects

  • Cell type-specific differences

• Evaluate kinetics: The timing of formylation relative to other cellular processes may be crucial:

  • Is formylation occurring before, during, or after nucleosome assembly?

  • Does the formylation persist or is it transient?

  • How does the kinetics of formylation compare with that of acetylation at the same site?

• Reexamine experimental controls: Based on findings that multiple lysine residues (K5, K8, K12) function redundantly , ensure your experimental design adequately controls for compensation by other sites. Consider:

  • Using combined mutations or modifications

  • Implementing acute rather than chronic interventions

  • Employing conditional systems to minimize adaptation

What statistical approaches are recommended for analyzing ChIP-seq data generated using Formyl-HIST1H4A (K12) antibody?

For robust statistical analysis of ChIP-seq data using Formyl-HIST1H4A (K12) antibody, implement this methodological framework:

• Quality control metrics:

  • Assess library complexity (PCR duplicates, unique fragment count)

  • Evaluate read mapping statistics (% mapped, % uniquely mapped)

  • Calculate signal-to-noise ratios (fraction of reads in peaks)

  • Perform cross-correlation analysis to verify enrichment

• Peak calling considerations:

  • Use appropriate peak calling algorithms (MACS2, SICER) with parameters adjusted for histone modifications

  • Implement stringent fold-enrichment thresholds (typically >4-fold over input)

  • Apply false discovery rate (FDR) correction (q < 0.05 or q < 0.01)

  • Consider broader domains rather than sharp peaks, as histone modifications often span regions

• Differential binding analysis:

  • Utilize specialized tools (DiffBind, MAnorm, or DESeq2 with appropriate normalization)

  • Apply batch effect correction if samples were processed in different batches

  • Consider quantile normalization to account for global differences in modification levels

• Biological replication:

  • Implement irreproducible discovery rate (IDR) analysis between replicates

  • Calculate Pearson correlation coefficients between normalized replicate profiles

  • Consider consensus peak sets requiring detection in multiple replicates

• Integration with other data types:

  • Correlate formylation patterns with transcriptome data

  • Compare with other histone modifications, particularly acetylation at K12

  • Analyze enrichment in different genomic annotations (promoters, enhancers, gene bodies)

How can researchers address potential artifacts in detection of H4K12 formylation during sample preparation?

Formylation can potentially occur as an artifact during sample preparation, particularly during fixation processes. To distinguish genuine biological formylation from technical artifacts:

• Modified fixation protocols:

  • Compare formaldehyde-fixed samples with alternative fixation methods

  • Implement gradient fixation protocols with varying formaldehyde concentrations

  • Test native (non-crosslinked) sample preparation when possible

• Chemical protection strategies:

  • Include formylation-quenching agents (e.g., glycine) during sample processing

  • Test the effect of antioxidants in buffers to prevent oxidative formylation

  • Use heavy isotope-labeled reagents to track potential artificial modifications

• Control samples for background estimation:

  • Process matched samples without fixation steps

  • Include negative controls from organisms or cell types known to lack the specific formylation

  • Implement spike-in normalization with standards containing known formylation levels

• Orthogonal validation:

  • Confirm findings using alternative detection methods (e.g., mass spectrometry)

  • Validate by manipulating enzymes responsible for formylation/deformylation

  • Compare results from multiple antibody clones or sources

How does the performance of Formyl-HIST1H4A (K12) antibody compare across different application techniques?

Based on available information, the Formyl-HIST1H4A (K12) antibody has been validated for ELISA, Western Blotting, Immunocytochemistry, and Immunofluorescence applications . When comparing performance across these techniques:

For optimal results across all applications, consider:

• Validating specificity using peptide competition assays

• Including appropriate positive and negative controls

• Optimizing blocking conditions to minimize background signal

• Testing multiple antibody concentrations to identify the optimal signal-to-noise ratio

What methodological differences should researchers consider when investigating formylation at H4K12 versus acetylation or other modifications?

When designing experiments to study formylation at H4K12 compared to other modifications like acetylation, implement these methodological considerations:

• Antibody selection and validation:

  • Confirm specificity against formylated vs. acetylated peptides

  • Test for cross-reactivity with other modified forms of H4K12

  • Consider the influence of neighboring modifications on epitope recognition

• Sample preparation differences:

  • Formylation may be more sensitive to oxidative conditions during extraction

  • Acetylation is affected by HDAC activity; consider HDAC inhibitors during sample preparation

  • Different fixation protocols may preferentially preserve certain modifications

• Experimental controls:

  • Include specific enzyme inhibitors:

    • For acetylation: HDAC inhibitors (e.g., TSA, sodium butyrate)

    • For formylation: Relevant enzyme inhibitors if known

  • Consider in vitro modified recombinant histones as standards

  • Compare wild-type samples with mutants where K12 is replaced with non-modifiable residues

• Data analysis considerations:

  • Formylation may have different genomic distribution patterns than acetylation

  • Temporal dynamics may differ (stability, enzyme kinetics)

  • Correlation with other histone marks may reveal distinct functional contexts

• Functional validation approaches:

  • Research on K5/K12 acetylation suggests redundancy with K8 in nucleosome assembly

  • Consider similar redundancy testing for formylation

  • Examine the effects under conditions where K5, K8, or other sites are also modified or mutated

When should researchers combine Formyl-HIST1H4A (K12) antibody with other techniques for comprehensive epigenetic analysis?

For comprehensive epigenetic analysis, the Formyl-HIST1H4A (K12) antibody should be combined with other techniques in these research scenarios:

• When establishing modification patterns across the genome:

  • Combine ChIP-seq using the Formyl-HIST1H4A (K12) antibody with similar analyses of:

    • Other histone modifications (particularly acetylation at K5, K8, K12, K16)

    • Histone variants

    • Chromatin accessibility (ATAC-seq, DNase-seq)

    • DNA methylation patterns

• When investigating functional consequences:

  • Correlate formylation patterns with:

    • Transcriptome data (RNA-seq)

    • Translation efficiency measurements

    • Protein-DNA interaction maps (other ChIP-seq data)

    • Chromosome conformation capture techniques (Hi-C, 4C)

• When studying temporal dynamics:

  • Implement time-course experiments combining:

    • ChIP-seq at multiple time points

    • Pulse-chase labeling of histones

    • Live-cell imaging with modification-specific sensors

    • Single-cell approaches to capture heterogeneity

• When analyzing mechanism:

  • Complement antibody-based detection with:

    • Mass spectrometry to identify co-occurring modifications

    • Enzyme activity assays for relevant writers/erasers

    • Protein-protein interaction studies to identify readers

    • CRISPR screens to identify regulatory factors

The research on H4 K5/K12 acetylation demonstrates that understanding the true biological function requires multiple complementary approaches, including both in vivo and in vitro assays, and consideration of potential redundancy with modifications at other sites .

How might the pattern of H4K12 formylation differ functionally from the well-established K5/K12 diacetylation pattern associated with histone deposition?

While newly synthesized histone H4 is typically diacetylated at K5/K12 in many organisms, research has shown that this specific pattern is not strictly required for nucleosome assembly in yeast . When investigating how H4K12 formylation might function differently:

• Contextual redundancy: Research has demonstrated that K5, K8, and K12 function redundantly in histone deposition, with assembly strongly impaired only when all three sites are mutated . This suggests that formylation at K12 might similarly participate in redundant regulatory systems, possibly serving as an alternative pathway when acetylation is compromised.

• Timing differences: Unlike acetylation, which is well-established as occurring on newly synthesized histones, formylation might occur post-incorporation into chromatin, potentially serving regulatory functions beyond deposition.

• Reader protein specificity: Different modifications recruit distinct reader proteins. While acetylation typically recruits bromodomain-containing proteins, formylation likely engages a different set of reader molecules, potentially activating distinct downstream pathways.

• Stability and reversibility: The enzymatic machinery for adding and removing formyl groups likely differs from that controlling acetylation, potentially resulting in different kinetic properties and response to cellular signals.

• Metabolic connections: Formylation may connect histone regulation to specific metabolic pathways distinct from those influencing acetylation, potentially serving as a mechanism to integrate metabolic state with chromatin structure.

Future research should explore these functional differences by comparing the genomic distribution, temporal dynamics, and protein interactions of formylated versus acetylated H4K12.

What experimental approaches would best elucidate whether H4K12 formylation participates in the redundancy observed with K5, K8, and K12 acetylation in nucleosome assembly?

Building on the finding that K5, K8, and K12 function redundantly in histone deposition , a comprehensive experimental strategy to investigate whether formylation at K12 participates in this redundancy would include:

• Mutation studies with formylation-specific controls:

  • Generate yeast strains with lysine-to-arginine (K→R) mutations that prevent acetylation but potentially allow formylation

  • Compare with lysine-to-glutamine (K→Q) mutations that mimic acetylation

  • Include controls that specifically prevent formylation while permitting acetylation

• In vivo nucleosome assembly assessment:

  • Measure plasmid superhelicity as an indicator of nucleosome formation in vivo

  • Compare assembly efficiency in strains with different mutation combinations

  • Analyze under conditions that promote or inhibit formylation

• In vitro reconstitution:

  • Perform nucleosome assembly assays using purified components

  • Compare assembly kinetics and stability using H4 with different modification states:

    • Unmodified at K5, K8, K12

    • Acetylated at various combinations of these sites

    • Formylated at K12 (with or without acetylation at K5/K8)

  • Analyze by native gel electrophoresis, MNase digestion patterns, and biophysical techniques

• Enzymatic manipulation:

  • Identify and manipulate enzymes responsible for formylation/deformylation

  • Assess the impact on nucleosome assembly when these enzymes are inhibited or overexpressed

  • Examine genetic interactions with known histone acetyltransferases/deacetylases

• Combined modification analysis:

  • Implement mass spectrometry approaches to quantify the co-occurrence of formylation and acetylation

  • Assess whether formylation increases when acetylation is blocked

  • Determine if there are conditions where formylation becomes critical for assembly

What are the emerging research directions regarding the potential relationship between histone H4K12 formylation and pathological conditions?

While direct evidence linking H4K12 formylation to pathological conditions is limited in the provided search results, several promising research directions emerge based on our understanding of histone modifications and disease:

• Neurodegenerative disorders:

  • Investigate H4K12 formylation patterns in models of Alzheimer's, Parkinson's, and other neurodegenerative diseases

  • Compare with known dysregulation of histone acetylation in these conditions

  • Assess correlation with cognitive deficits and disease progression

• Cancer epigenetics:

  • Profile H4K12 formylation across cancer types and stages

  • Correlate patterns with oncogene expression and tumor suppressor silencing

  • Explore potential as a biomarker for specific cancer subtypes or progression states

• Inflammatory conditions:

  • Examine changes in H4K12 formylation during acute and chronic inflammation

  • Investigate connection to oxidative stress, which can promote formylation

  • Assess relationship with inflammatory gene expression programs

• Metabolic disorders:

  • Study how metabolic perturbations affect H4K12 formylation levels

  • Investigate in models of diabetes, obesity, and metabolic syndrome

  • Explore connection to mitochondrial dysfunction and altered cellular metabolism

• Therapeutic targeting:

  • Identify enzymes responsible for adding/removing formyl groups

  • Develop small molecule inhibitors or activators of these enzymes

  • Assess therapeutic potential in relevant disease models

Future studies should employ advanced techniques such as genome-wide profiling of H4K12 formylation in patient samples, CRISPR-mediated manipulation of formylation levels, and development of specific inhibitors/activators for formylation regulatory enzymes.