Formyl-HIST1H1E (K62) Antibody

What are the primary research applications for Formyl-HIST1H1E (K62) antibodies?

Formyl-HIST1H1E (K62) antibodies are valuable tools for:

• Chromatin immunoprecipitation (ChIP) experiments to identify genomic binding sites of formylated HIST1H1E

• Immunofluorescence imaging to visualize nuclear distribution patterns

• Western blotting to quantify formylation levels across different cell types and conditions

• Investigating epigenetic modifications in neurodevelopmental disorders, particularly in Rahman syndrome models

• Examining temporal dynamics of histone modifications during cellular differentiation and stress response

How do mutations in HIST1H1E relate to potential changes in formylation patterns?

Frameshift mutations in HIST1H1E, particularly those affecting the C-terminal domain, have been associated with Rahman syndrome, characterized by intellectual disability and distinctive facial features . These mutations may potentially alter post-translational modification patterns, including formylation at K62. Researchers investigating the molecular pathogenesis of HIST1H1E-related disorders should consider how these mutations might affect formylation status, potentially disrupting chromatin organization and gene expression patterns during neurodevelopment.

What are the optimal fixation and sample preparation protocols for Formyl-HIST1H1E (K62) antibody applications?

For optimal results with Formyl-HIST1H1E (K62) antibody applications:

Immunohistochemistry/Immunofluorescence Preparation:

• Use freshly prepared 4% paraformaldehyde for tissue fixation (15-20 minutes)

• Perform antigen retrieval using citrate buffer (pH 6.0) at 95-100°C for 15-20 minutes

• Include a permeabilization step with 0.2% Triton X-100 for 10 minutes

• Block with 3-5% BSA or normal serum for at least 1 hour

• Incubate with primary antibody at optimal dilution (typically 1:100-1:500) at 4°C overnight

Western Blot Sample Preparation:

• Extract histones using specialized acid extraction protocols to efficiently recover histone proteins

• Include deacetylase and demethylase inhibitors (e.g., sodium butyrate, nicotinamide) in lysis buffers

• Maintain samples at 4°C throughout processing to prevent degradation of post-translational modifications

How can researchers validate specificity of Formyl-HIST1H1E (K62) antibody signals?

Antibody validation is critical for ensuring experimental reliability. Recommended approaches include:

• Peptide Competition Assays: Pre-incubate antibody with formylated and non-formylated HIST1H1E peptides containing the K62 residue

• Knockout/Knockdown Controls: Compare signals in wild-type versus HIST1H1E-depleted samples

• Modification-Specific Controls: Compare signals after treatment with deformylase enzymes

• Cross-Reactivity Testing: Evaluate potential binding to other formylated histones, particularly other H1 variants

• Mass Spectrometry Validation: Confirm formylation at K62 in immunoprecipitated samples

What are the recommended protocols for ChIP applications with Formyl-HIST1H1E (K62) antibodies?

For optimal ChIP results with Formyl-HIST1H1E (K62) antibodies:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize conditions to generate chromatin fragments of 200-500 bp

• Immunoprecipitation:

  • Use 2-5 μg antibody per chromatin sample (derived from ~1-2 × 10^6 cells)

  • Include appropriate controls (IgG, input DNA, non-formylated HIST1H1E)

  • Incubate overnight at 4°C with gentle rotation

• Washing: Perform stringent washing steps to reduce background (at least 5 washes)

• Elution and Reversal: Elute immunocomplexes and reverse crosslinks at 65°C for 4-6 hours

• DNA Purification: Use silica column-based methods for optimal DNA recovery

• Analysis: Perform qPCR or next-generation sequencing to analyze enriched genomic regions

How can researchers differentiate between formylation and other lysine modifications at the K62 position?

Distinguishing formylation from other lysine modifications (acetylation, methylation) requires specialized approaches:

• Mass Spectrometry Analysis:

  • Use high-resolution MS/MS to identify the precise mass shift associated with formylation (+28 Da)

  • Implement targeted approaches focusing on K62-containing peptides

  • Apply multiple fragmentation techniques (CID, ETD, HCD) for comprehensive analysis

• Combinatorial Antibody Approaches:

  • Use antibodies specific for different modifications at K62 in parallel experiments

  • Perform sequential immunoprecipitation to identify co-occurrence of modifications

• Enzymatic Treatment Controls:

  • Compare immunoreactivity after treatment with deformylases versus deacetylases

  • Use recombinant proteins with defined modifications as positive controls

What are the technical challenges in studying Formyl-HIST1H1E in patient-derived samples with HIST1H1E mutations?

Studying formylation in patient samples presents several technical challenges:

• Limited Sample Availability: Rahman syndrome is rare, with only about 52 documented patients exhibiting HIST1H1E variants

• Variant-Specific Effects: Different frameshift mutations may differentially impact post-translational modification patterns

• Tissue-Specific Considerations:

  • Neural tissues are challenging to access in living patients

  • Peripheral blood may not reflect neural epigenetic patterns

  • Patient-derived iPSCs may be required for neuronal differentiation models

• Detection Sensitivity:

  • Formylation levels may be low and technically challenging to detect

  • Background signal may complicate interpretation in heterozygous mutation carriers

• Methodological Solutions:

  • Use highly sensitive techniques like CUT&RUN or CUT&Tag

  • Implement single-cell approaches to address cellular heterogeneity

  • Develop patient-derived organoid models for more physiologically relevant analyses

How can formylation patterns at HIST1H1E (K62) be correlated with chromatin accessibility and gene expression?

To establish correlations between formylation, chromatin accessibility, and gene expression, researchers should implement multi-omic approaches:

• Integrated Analysis Pipeline:

  • Perform ChIP-seq with Formyl-HIST1H1E (K62) antibodies

  • Couple with ATAC-seq to measure chromatin accessibility

  • Integrate RNA-seq data to correlate with gene expression patterns

  • Include Hi-C or other chromosome conformation capture techniques

• Bioinformatic Analysis Strategies:

  • Identify genomic regions enriched for formylated HIST1H1E

  • Compare with accessibility peaks from ATAC-seq

  • Correlate with differentially expressed genes

  • Apply machine learning approaches to identify predictive patterns

• Temporal Analysis:

  • Track changes during developmental processes

  • Monitor response to environmental stimuli

  • Examine cell cycle-dependent fluctuations

How does HIST1H1E formylation at K62 potentially relate to the pathogenesis of Rahman syndrome?

Rahman syndrome, caused by frameshift mutations in HIST1H1E, presents with intellectual disability, distinctive facial features, and other neurodevelopmental abnormalities . The relationship between K62 formylation and disease pathogenesis may involve:

• Altered Chromatin Compaction: Formylation may influence histone-DNA binding affinity, potentially disrupting normal chromatin architecture

• Dysregulated Gene Expression: Changes in chromatin structure could lead to aberrant gene expression patterns during critical neurodevelopmental windows

• Developmental Consequences: The 23 frameshift variants identified in 52 patients all result in almost identical shorter proteins with a shared divergent C-terminal tail , which may alter the pattern or recognition of post-translational modifications like formylation

• Cellular Senescence Effects: Some HIST1H1E mutations accelerate cellular senescence and cause premature aging phenotypes , which might be linked to changes in formylation patterns

What are the best model systems for studying Formyl-HIST1H1E (K62) in the context of neurodevelopmental disorders?

Several model systems offer complementary advantages for studying Formyl-HIST1H1E (K62) in neurodevelopmental contexts:

Cell-Based Models:

• iPSC-derived neuronal cultures from patient samples

• CRISPR-engineered cell lines with specific HIST1H1E mutations

• Neural organoids that recapitulate aspects of brain development

Animal Models:

• Mouse models with equivalent mutations in the murine Hist1h1e gene

• Conditional knockout/knockin models for temporal and tissue-specific studies

Experimental Design Considerations:

• Include developmental time-course analyses

• Examine multiple neural cell types (neurons, glia, neural progenitors)

• Compare heterozygous versus homozygous mutations to assess dose-dependent effects

• Implement rescue experiments with wild-type HIST1H1E

How can researchers distinguish pathogenic effects of altered formylation from other consequences of HIST1H1E mutations?

Distinguishing specific effects of altered formylation from other mutation consequences requires sophisticated experimental approaches:

• Site-Specific Mutation Analysis:

  • Generate K62R mutants (preventing formylation) without affecting other protein functions

  • Compare with disease-causing frameshift mutations

  • Assess differential effects on chromatin structure and gene expression

• Enzymatic Modulation Experiments:

  • Manipulate formylation enzymes while maintaining HIST1H1E sequence

  • Use small molecule inhibitors or activators of formylation pathways

  • Compare phenotypic outcomes with mutation models

• Modification-Specific Protein Interactions:

  • Identify protein complexes that specifically recognize formylated K62

  • Determine if these interactions are disrupted in disease models

  • Assess downstream signaling and transcriptional consequences

What technological advances might improve detection and quantification of Formyl-HIST1H1E (K62)?

Emerging technologies with potential to advance Formyl-HIST1H1E (K62) research include:

• Advanced Antibody Technologies:

  • Single-domain antibodies with improved specificity

  • Recombinant antibody fragments with enhanced epitope recognition

  • Modification-specific nanobodies for live-cell imaging

• Novel Microscopy Approaches:

  • Super-resolution microscopy to visualize chromatin organization

  • Live-cell imaging of formylation dynamics

  • Correlative light and electron microscopy for ultrastructural context

• Mass Spectrometry Innovations:

  • Improved sensitivity for detecting low-abundance modifications

  • Quantitative approaches for precise measurement of formylation stoichiometry

  • Single-cell proteomics for heterogeneity analysis

How might multi-omics approaches enhance understanding of HIST1H1E formylation in chromatin regulation?

Integration of multiple omics technologies offers comprehensive insights into formylation biology:

• Complementary Technologies:

  • ChIP-seq: Genomic localization of formylated HIST1H1E

  • ATAC-seq: Chromatin accessibility correlations

  • CUT&RUN/CUT&Tag: Higher resolution mapping of histone modifications

  • Hi-C/Micro-C: 3D chromatin architecture analysis

  • RNA-seq: Transcriptional consequences

  • Proteomics: Interaction partners and co-occurring modifications

• Integrated Analysis Frameworks:

  • Computational methods to correlate data across platforms

  • Machine learning approaches to identify predictive patterns

  • Network analysis to map regulatory relationships

• Single-Cell Multi-Omics:

  • Combined measurement of chromatin, transcription, and protein states

  • Cellular heterogeneity assessment

  • Trajectory analysis during differentiation or disease progression