2-hydroxyisobutyryl-HIST1H1C (K63) Antibody

What is 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody and what epitope does it recognize?

2-hydroxyisobutyryl-HIST1H1C (K63) Antibody is a polyclonal antibody that specifically recognizes the 2-hydroxyisobutyrylation modification at lysine 63 (K63) of human Histone H1.2 protein (also known as HIST1H1C). The antibody is raised in rabbits against a peptide sequence surrounding the 2-hydroxyisobutyryl-lysine 63 site derived from Human Histone H1.2 . This post-translational modification is part of the expanding "histone code" that regulates chromatin structure and gene expression.

The antibody's immunogen consists of a synthetic peptide containing the modified amino acid sequence surrounding the K63 position of HIST1H1C. This site-specific recognition makes the antibody a valuable tool for studying this particular histone modification in epigenetic research .

What applications is the 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody validated for?

The 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody has been validated for several laboratory applications:

For immunofluorescence applications, the recommended dilution range is 1:50 to 1:200, but optimal dilutions should be determined by the researcher based on their specific experimental conditions .

Unlike some other 2-hydroxyisobutyryl-HIST1H1C antibodies targeting different lysine residues (such as K158), this particular antibody has not been explicitly validated for Western blot (WB), Immunocytochemistry (ICC), or Chromatin Immunoprecipitation (ChIP) applications according to the available data .

What is the functional significance of 2-hydroxyisobutyrylation at K63 of HIST1H1C?

2-hydroxyisobutyrylation is a relatively recently discovered histone post-translational modification that plays important roles in epigenetic regulation. The modification at K63 of HIST1H1C (Histone H1.2) is particularly significant because:

• Histone H1.2 binds to linker DNA between nucleosomes, forming the chromatin fiber structure

• It participates in the condensation of nucleosome chains into higher-order structured fibers

• It functions as a regulator of gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation

The specific 2-hydroxyisobutyrylation at K63 likely contributes to these functions by altering the binding properties of HIST1H1C or by recruiting specific reader proteins that recognize this modification. The modification may also influence the interaction of HIST1H1C with linker DNA or with other proteins involved in chromatin organization.

How should the 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody be stored and handled?

For optimal performance and longevity of the 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody, proper storage and handling are essential:

• Upon receipt, store the antibody at -20°C or -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles as they can degrade the antibody and reduce its effectiveness

• The antibody is provided in liquid form containing preservative (0.03% Proclin 300) and buffer constituents (50% Glycerol, 0.01M PBS, pH 7.4)

• When working with the antibody, keep it on ice and return to storage promptly after use

• For diluted working solutions, prepare fresh for each experiment or store small aliquots at -20°C for short periods

Proper storage conditions are critical for maintaining antibody specificity and activity, particularly for antibodies recognizing post-translational modifications such as 2-hydroxyisobutyrylation.

How can I optimize immunofluorescence experiments using 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody?

Optimizing immunofluorescence (IF) experiments with 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody requires attention to several key factors:

• Fixation method: For optimal detection of histone modifications, paraformaldehyde fixation (4%) for 10-15 minutes at room temperature is generally recommended. Methanol fixation may cause loss of some histone modifications.

• Permeabilization: Use 0.1-0.5% Triton X-100 in PBS for 5-10 minutes to ensure antibody access to nuclear antigens.

• Blocking conditions: Block with 1-5% BSA or normal serum (from the species of the secondary antibody) in PBS for 30-60 minutes to reduce background.

• Antibody dilution: Start with the recommended range (1:50-1:200) , but perform a dilution series to determine optimal concentration for your specific cell type and conditions.

• Incubation time and temperature: For primary antibody, incubate overnight at 4°C or 1-2 hours at room temperature. For secondary antibody, 1 hour at room temperature is typically sufficient.

• Controls: Include:

  • No primary antibody control

  • Isotype control

  • Peptide competition control using the unmodified K63 peptide

  • Positive control (cell type known to express high levels of 2-hydroxyisobutyrylated HIST1H1C)

• Signal amplification: For weak signals, consider using tyramide signal amplification or high-sensitivity detection systems.

• Counterstaining: Include DAPI or Hoechst staining to visualize nuclei and confirm nuclear localization of the signal.

After optimization, document all parameters carefully to ensure reproducibility across experiments.

How does 2-hydroxyisobutyrylation at K63 differ from other lysine modifications on HIST1H1C?

HIST1H1C (Histone H1.2) undergoes various post-translational modifications at multiple lysine residues. The 2-hydroxyisobutyrylation at K63 differs from other modifications in several important aspects:

The K63 residue is positioned within the globular domain of HIST1H1C, which directly interacts with nucleosomal DNA. 2-hydroxyisobutyrylation at this position likely affects:

• The binding affinity of HIST1H1C to nucleosomal DNA

• The structural conformation of the globular domain

• Interaction with other chromatin-associated proteins

• Accessibility of DNA to transcription factors

Unlike acetylation (which neutralizes the positive charge of lysine) and methylation (which preserves the charge), 2-hydroxyisobutyrylation adds a bulkier group that could create more significant steric effects while also neutralizing the charge. This may result in more dramatic changes to chromatin accessibility at K63-modified sites compared to other modifications at the same position .

What experimental approaches can validate the specificity of 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody?

Validating antibody specificity is crucial, especially for post-translational modification-specific antibodies. For 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody, consider these approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with:

    • The 2-hydroxyisobutyrylated K63 peptide immunogen (should eliminate signal)

    • Unmodified K63 peptide (should not affect signal)

    • 2-hydroxyisobutyrylated peptides from other lysine residues of HIST1H1C (minimal effect if antibody is specific)

• Knockout/knockdown validation:

  • Use HIST1H1C knockout or knockdown cells to verify signal disappearance

  • For complete validation, use cells with K63R mutation (prevents modification)

• Mass spectrometry correlation:

  • Compare antibody-based detection (IF/WB) with mass spectrometry quantification of the modification

  • Examine concordance across different experimental conditions

• Cross-reactivity testing:

  • Test against other 2-hydroxyisobutyrylated histones (H2A, H2B, H3, H4)

  • Test against HIST1H1C with other modifications at K63 (acetylation, methylation)

• Dot blot analysis:

  • Create a panel of modified and unmodified peptides on a membrane

  • Probe with the antibody to assess specific binding

• Parallel antibody comparison:

  • Compare results with alternate antibodies targeting the same modification (if available)

  • Different clones should give similar results if specific

Documenting these validation experiments is essential for publication and ensures reliable interpretation of experimental results.

How do cell type and physiological conditions affect 2-hydroxyisobutyrylation patterns at HIST1H1C K63?

2-hydroxyisobutyrylation at HIST1H1C K63 can vary significantly based on cell type and physiological conditions:

• Cell cycle dependence:

  • During S phase: Increased 2-hydroxyisobutyrylation often occurs to facilitate chromatin accessibility for DNA replication

  • During mitosis: Modified patterns align with chromosome condensation requirements

• Metabolic influences:

  • 2-hydroxyisobutyrylation requires 2-hydroxyisobutyrate as a substrate

  • Cellular metabolic state affects substrate availability

  • Glycolytic vs. oxidative phosphorylation predominance may shift modification levels

• Cell type variability:

  • Stem cells: Often show distinct patterns supporting pluripotency

  • Differentiated cells: Pattern reflects tissue-specific gene expression needs

  • Cancer cells: Frequently display aberrant modification patterns

• Stress responses:

  • Oxidative stress: Can alter enzymatic activity of writers/erasers of this modification

  • Nutrient deprivation: Affects metabolite availability for modification

  • DNA damage: May trigger specific modification patterns for repair processes

• Developmental changes:

  • Embryonic vs. adult tissues show different modification landscapes

  • Cell differentiation involves programmed changes in histone modifications

When designing experiments, researchers should:

• Consider time course studies to capture dynamic changes

• Standardize culture conditions carefully

• Document cell confluence and passage number

• Control for metabolic variables (media composition, serum levels)

• Validate findings across multiple cell lines or primary cells

These considerations are crucial for reproducibility and meaningful interpretation of 2-hydroxyisobutyrylation data.

How can 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody be used in combination with other techniques to study chromatin dynamics?

Combining 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody with other techniques creates powerful approaches for studying chromatin dynamics:

• ChIP-seq integration:

  • While not explicitly validated for ChIP, optimization may enable:

    • Genome-wide mapping of K63 2-hydroxyisobutyrylation

    • Integration with transcriptomic data to correlate with gene expression

    • Comparison with other histone modifications for co-occurrence patterns

• Mass spectrometry pairing:

  • Immunoprecipitate with the antibody followed by MS analysis to:

    • Identify proteins that interact with 2-hydroxyisobutyryl-K63 HIST1H1C

    • Quantify modification stoichiometry at K63 vs. other sites

    • Discover co-occurring modifications on the same histone molecule

• Super-resolution microscopy:

  • Use the antibody for immunofluorescence combined with techniques like:

    • STORM/PALM for nanoscale localization

    • Live-cell imaging with tagged reader proteins to track dynamics

    • Correlative light and electron microscopy for structural context

• Proximity ligation assays:

  • Detect interactions between 2-hydroxyisobutyryl-K63 HIST1H1C and:

    • Transcription factors

    • Chromatin remodelers

    • Other histone-modifying enzymes

• ATAC-seq or DNase-seq correlation:

  • Compare 2-hydroxyisobutyryl-K63 HIST1H1C localization with:

    • Chromatin accessibility profiles

    • Nucleosome positioning data

    • DNA methylation patterns

• Writer/eraser enzyme manipulation:

  • Combine antibody detection with:

    • Overexpression/knockdown of suspected enzymes

    • Chemical inhibitors of modifying enzymes

    • Metabolic manipulation of substrate availability

For data integration, computational approaches are essential:

• Multivariate analysis of different epigenetic marks

• Machine learning to identify patterns and predictive features

• Network analysis to understand modification hierarchies

These integrated approaches provide much deeper insights than any single technique alone.

What are common issues when using 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody and how can they be resolved?

Researchers may encounter several technical challenges when working with 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody. Here are common issues and their solutions:

When troubleshooting, it's advisable to:

• Initially test the antibody under standard conditions recommended by the manufacturer

• Change only one parameter at a time

• Document all experimental conditions carefully

• Consult technical support from the antibody provider

• Consider antibody validation services if persistent issues occur

How should data from 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody experiments be quantified and statistically analyzed?

For Immunofluorescence Data:

• Image acquisition standardization:

  • Use identical exposure settings across all conditions

  • Collect multiple fields of view (minimum 5-10 per condition)

  • Image at least 50-100 cells per condition

• Quantification approaches:

  • Nuclear intensity measurements:

    • Mean fluorescence intensity (MFI)

    • Integrated density (area × mean intensity)

  • Subnuclear distribution:

    • Coefficient of variation of nuclear staining

    • Co-localization with heterochromatin/euchromatin markers

• Normalization strategies:

  • To DAPI intensity (controls for DNA content)

  • To total HIST1H1C (using a non-modification-specific antibody)

  • To nuclear area

Statistical Analysis:

Researchers should always report:

• Number of biological and technical replicates

• Specific statistical tests used with justification

• P-values or confidence intervals

• Effect sizes, not just statistical significance

How can 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody be used to study disease mechanisms?

The 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody offers valuable approaches for investigating disease mechanisms, particularly in conditions involving epigenetic dysregulation:

• Cancer research applications:

  • Compare 2-hydroxyisobutyrylation patterns between:

    • Tumor vs. adjacent normal tissue

    • Different cancer stages/grades

    • Treatment-responsive vs. resistant tumors

  • Correlate modification levels with:

    • Oncogene expression

    • Tumor suppressor silencing

    • Patient outcomes

  • Target for potential epigenetic therapy development

• Neurodegenerative disorders:

  • Examine age-related changes in 2-hydroxyisobutyrylation

  • Study modification patterns in models of Alzheimer's, Parkinson's, etc.

  • Investigate interaction with disease-specific protein aggregates

• Metabolic disorders:

  • Link cellular metabolism to epigenetic regulation

  • Study how metabolic stress affects 2-hydroxyisobutyrylation

  • Examine modification changes in diabetes, obesity models

• Inflammatory conditions:

  • Examine dynamic changes during inflammatory responses

  • Study modification patterns in immune cells during activation

  • Investigate role in inflammatory gene regulation

• Developmental disorders:

  • Compare modification patterns in normal vs. abnormal development

  • Study role in cell differentiation and lineage commitment

  • Examine transgenerational epigenetic inheritance

Research approaches could include:

• Patient-derived samples (tissue microarrays, biopsies)

• Disease-relevant cell culture models

• Animal models of human diseases

• Drug screening to identify compounds affecting this modification

This antibody enables researchers to uncover how this specific histone modification contributes to disease pathogenesis and potentially identify new therapeutic targets.

What is the relationship between 2-hydroxyisobutyrylation at K63 and other HIST1H1C modifications in the context of the histone code?

The 2-hydroxyisobutyrylation at K63 of HIST1H1C operates within a complex network of histone modifications that constitute the "histone code." Understanding these relationships provides deeper insights into chromatin regulation:

• Sequential modification patterns:

  • Priming effects: Some modifications must precede others

    • Phosphorylation at neighboring sites may facilitate or inhibit K63 2-hydroxyisobutyrylation

    • Methylation and 2-hydroxyisobutyrylation are typically mutually exclusive at the same lysine

• Cross-talk with other HIST1H1C modifications:

• Writer/eraser/reader interactions:

  • Many enzymes that modify histones work in complexes

  • 2-hydroxyisobutyrylation at K63 may recruit specific reader proteins

  • These readers can then recruit additional modifying enzymes

  • Creates feedback loops and modification cascades

• Functional outcomes of combined modifications:

  • Additive effects: Multiple modifications reinforcing same outcome

  • Antagonistic effects: One modification counteracting another

  • Contextual effects: Outcome depends on additional modifications present

• Technological approaches to study these relationships:

  • Middle-down mass spectrometry to identify co-occurring modifications

  • Sequential ChIP (re-ChIP) to identify co-localization on same DNA regions

  • Combinatorial antibody approaches (e.g., proximity ligation assays)

  • CRISPR-based editing of specific residues to test dependencies

Understanding these relationships is critical for deciphering the complex language of histone modifications and their role in gene regulation.

How do emerging single-cell techniques impact research using 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody?

Single-cell technologies are revolutionizing epigenetic research and offer new opportunities for studies using 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody:

• Single-cell epigenomic approaches:

  • Single-cell CUT&Tag/CUT&RUN:

    • Could be adapted for 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody

    • Maps modification genome-wide in individual cells

    • Reveals cell-to-cell heterogeneity masked in bulk analyses

  • Single-cell immunofluorescence:

    • High-content imaging with the antibody

    • Quantifies modification levels in thousands of individual cells

    • Can be combined with other markers for multiparametric analysis

  • Mass cytometry (CyTOF):

    • Metal-tagged antibodies for high-dimensional analysis

    • Can simultaneously measure multiple histone modifications

    • Requires metal conjugation of the antibody

• Integration with other single-cell data types:

  • Multimodal approaches combining:

    • 2-hydroxyisobutyryl-HIST1H1C (K63) detection

    • Single-cell transcriptomics

    • Chromatin accessibility (scATAC-seq)

    • DNA methylation

• Technical considerations for single-cell applications:

  • Higher antibody concentrations often needed for single-cell techniques

  • Fixation protocols must balance antigen preservation with cell integrity

  • Signal amplification strategies become more critical

  • Stringent validation needed to ensure specificity at single-cell level

• Data analysis challenges:

  • Computational integration of sparse single-cell data

  • Trajectory analysis to map modification changes during cell state transitions

  • Machine learning for pattern recognition across thousands of cells

  • Spatial analysis when combining with imaging approaches

• Emerging applications:

  • Mapping epigenetic heterogeneity in tumor microenvironments

  • Tracking dynamic modification changes during differentiation

  • Identifying rare cell populations with distinct epigenetic states

  • Spatial epigenomics using in situ detection methods

These approaches will provide unprecedented resolution of how 2-hydroxyisobutyrylation at K63 of HIST1H1C varies between individual cells within populations, revealing functional heterogeneity previously masked in bulk analyses.

What are the current limitations in 2-hydroxyisobutyryl-HIST1H1C (K63) research and future directions?

Current research on 2-hydroxyisobutyryl-HIST1H1C (K63) faces several limitations that simultaneously point to important future research directions:

• Technical limitations:

  • Limited validation of antibodies across all applications (particularly ChIP)

  • Challenges in quantifying modification stoichiometry

  • Difficulty distinguishing cellular heterogeneity from technical variability

  • Incomplete understanding of writer/eraser enzymes specific for K63

• Knowledge gaps:

  • Unknown reader proteins that specifically recognize this modification

  • Incomplete understanding of crosstalk with other histone modifications

  • Limited information on tissue-specific patterns

  • Unclear role in disease pathogenesis

  • Unknown metabolic pathways that regulate substrate availability

• Future research directions:

  • Enzymatic regulation:

    • Identification of site-specific writers and erasers for K63

    • Structural studies of enzyme-substrate interactions

    • Development of small molecule modulators

  • Functional studies:

    • CRISPR-based K63R mutations to assess biological significance

    • Identification of reader proteins using proteomics approaches

    • Mechanistic studies linking modification to chromatin structure changes

  • Technological advances:

    • Development of reader domain-based biosensors for live imaging

    • Improved antibodies with increased specificity and affinity

    • New chemical biology tools to manipulate the modification

    • Integrated multi-omics approaches

  • Translational potential:

    • Biomarker development for disease states

    • Therapeutic targeting of regulatory enzymes

    • Nutritional interventions affecting substrate availability

• Methodological improvements needed:

  • Standardized protocols for detection and quantification

  • Reference materials for antibody validation

  • Improved computational tools for data integration

  • Community standards for reporting modification data

These advances will require interdisciplinary collaboration between epigenetics researchers, structural biologists, analytical chemists, and computational scientists to fully understand the biological significance of 2-hydroxyisobutyrylation at K63 of HIST1H1C.

How does understanding 2-hydroxyisobutyryl-HIST1H1C (K63) contribute to the broader field of epigenetics?

Research on 2-hydroxyisobutyryl-HIST1H1C (K63) contributes significantly to the broader field of epigenetics in several important ways:

• Expanding the histone code:

  • 2-hydroxyisobutyrylation represents a relatively new addition to known histone modifications

  • Understanding site-specific effects at K63 helps refine the histone code hypothesis

  • Demonstrates the complexity and specificity of histone modifications beyond the classic acetylation and methylation marks

• Linking metabolism to epigenetic regulation:

  • 2-hydroxyisobutyrylation directly connects cellular metabolic state to chromatin structure

  • Positions linker histones as metabolic sensors affecting higher-order chromatin organization

  • Supports the emerging field of metabolo-epigenetics

• Redefining linker histone function:

  • Traditionally viewed as structural proteins, this research highlights their regulatory roles

  • Shows how post-translational modifications fine-tune H1 function

  • Challenges the conventional view of linker histones as mere chromatin compactors

• Methodological advancements:

  • Drives development of site-specific antibodies for histone modifications

  • Encourages refinement of analytical techniques for studying chromatin biology

  • Promotes interdisciplinary approaches combining genomics, proteomics, and imaging

• Evolutionary insights:

  • Conservation of modification sites across species informs understanding of fundamental regulatory mechanisms

  • Variations between species highlight adaptive chromatin regulatory mechanisms

  • Provides insights into the evolution of epigenetic complexity

• Therapeutic implications:

  • Identifies potential new targets for epigenetic therapies

  • Supports precision medicine approaches targeting specific modifications

  • May reveal novel biomarkers for disease diagnosis and prognosis

By advancing our understanding of 2-hydroxyisobutyryl-HIST1H1C (K63), researchers contribute to the larger framework of how epigenetic mechanisms control gene expression and cellular function, with implications ranging from basic chromatin biology to human disease treatment.

What key resources are available for researchers working with 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody?

Researchers working with 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody have access to several valuable resources:

• Commercial antibodies and related products:

  • AFG Scientific offers 2-hydroxyisobutyryl-HIST1H1C (K63) Antibody (SKU: A24722) in 50ul and 100ul sizes

  • Related antibodies targeting other 2-hydroxyisobutyrylation sites on HIST1H1C are available (K109, K116, K128, K135, K158, K168, K210, K22, K25, K26, K51)

  • Complementary antibodies against total HIST1H1C (non-modification specific) are available from suppliers like Abcam (ab17677)

• Protein and sequence resources:

  • UniProtKB entry for human HIST1H1C: P16403

  • Sequence information helps identify the context of the K63 residue

  • Post-translational modification databases like PhosphoSitePlus

• Reference materials and protocols:

  • Manufacturer's datasheets provide specific application protocols

  • Methods papers on histone modification detection

  • ChIP-seq and immunofluorescence protocol repositories

• Validation resources:

  • Synthetic modified peptides for antibody validation

  • CRISPR-engineered cell lines with K-to-R mutations

  • Mass spectrometry services for confirmation of modifications

• Bioinformatics tools:

  • Histone modification databases

  • Chromatin state prediction algorithms

  • Data integration platforms for multi-omics analysis

  • Visualization tools for epigenomic data

• Research community resources:

  • Epigenetics-focused scientific conferences

  • Field-specific journals with methodological focuses

  • Online forums and communities for technical troubleshooting

  • Collaborative research networks and consortia

When using these resources, researchers should:

• Keep detailed records of lot numbers and validation experiments

• Share protocols and validation data with the research community

• Contribute to antibody validation databases where available

• Consider publishing methodological advancements as research resources