2-hydroxyisobutyryl-HIST1H3A (K36) Antibody

What is 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody and what does it detect?

The 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody is a rabbit polyclonal antibody that specifically recognizes the 2-hydroxyisobutyrylation post-translational modification at lysine 36 of histone H3.1 (HIST1H3A). This antibody was generated using a synthesized peptide derived from human Histone H3.1 protein (amino acids 29-40) containing the modified K36 residue . As a polyclonal IgG antibody, it binds specifically to the peptide sequence surrounding this modified lysine, making it a valuable tool for studying this relatively novel histone modification in epigenetic research contexts.

What are the validated applications for this antibody?

According to manufacturer specifications, the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody has been validated for ELISA (Enzyme-Linked Immunosorbent Assay) and IF/ICC (Immunofluorescence/Immunocytochemistry) applications . For immunofluorescence applications, the recommended dilution range is 1:50-1:200 . These applications enable researchers to detect and quantify the presence and distribution of 2-hydroxyisobutyrylation at H3K36 in various biological contexts, from measuring global modification levels to visualizing nuclear localization patterns.

How does this antibody differ from other histone modification antibodies?

Unlike antibodies targeting more well-characterized histone modifications such as methylation or acetylation, the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody recognizes a relatively newly discovered post-translational modification. This antibody specifically targets the 2-hydroxyisobutyrylation at lysine 36 of histone H3.1, which is structurally distinct from other acylation modifications like acetylation. While antibodies detecting H3K36 methylation (particularly trimethylation) have been widely used to study transcription elongation , this antibody provides researchers with a tool to investigate a different modification at the same residue, potentially revealing complementary or distinct functions in transcriptional regulation.

What are the optimal storage conditions for maintaining antibody activity?

For maximum preservation of antibody functionality, the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody should be stored at -20°C or -80°C upon receipt . The antibody is supplied in liquid form in a buffer containing preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . It is critically important to avoid repeated freeze/thaw cycles as they can lead to protein denaturation and gradual loss of antibody activity . For practical laboratory use, it is recommended to prepare small working aliquots of the antibody before freezing to minimize the number of freeze/thaw cycles each aliquot experiences.

What is the verified species reactivity of this antibody?

According to the product specifications, the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody has been specifically verified to react with human samples . This is consistent with its development using a human histone H3.1-derived immunogen. While cross-reactivity with other species may be possible due to the high conservation of histone proteins across evolution, experimental validation would be necessary before using this antibody for non-human applications.

What controls should be included when using this antibody for immunofluorescence?

When designing immunofluorescence experiments with the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody, several critical controls should be included:

• Negative controls:

  • Isotype control: Include a sample stained with non-specific rabbit IgG at the same concentration as the primary antibody

  • Omission control: Process a sample without primary antibody to assess secondary antibody background

  • Peptide competition: Pre-incubate the antibody with excess immunizing peptide to demonstrate binding specificity

• Positive controls:

  • Cell types known to exhibit high levels of histone 2-hydroxyisobutyrylation

  • Treatment controls (e.g., cells treated with HDAC inhibitors that may increase modification levels)

• Technical controls:

  • Nuclear counterstain (DAPI or Hoechst) to confirm nuclear localization

  • Additional histone modification antibodies to establish staining patterns for comparison

These controls collectively establish the specificity and reliability of the observed staining patterns.

How should I optimize the fixation protocol for detecting this modification?

Optimal detection of histone modifications requires careful consideration of fixation conditions to preserve both nuclear architecture and epitope accessibility. For the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody, consider the following fixation approach:

• Paraformaldehyde fixation: Use freshly prepared 4% paraformaldehyde in PBS for 10-15 minutes at room temperature. This provides good nuclear structure preservation while maintaining accessibility to nuclear antigens.

• Alternative approach: A sequential fixation with 2% formaldehyde for 10 minutes followed by ice-cold methanol for 5 minutes can provide excellent results for nuclear histone modifications.

• Avoid overfixation: Extended fixation times or higher concentrations of fixatives can lead to excessive cross-linking and epitope masking.

• Permeabilization: Following fixation, permeabilize cells with 0.1-0.2% Triton X-100 in PBS for 5-10 minutes to facilitate antibody access to nuclear antigens.

• Epitope retrieval: If signal is weak after standard fixation, consider mild antigen retrieval using 10mM citrate buffer (pH 6.0) at 95°C for 10 minutes.

Optimization may be required for specific cell types or experimental conditions.

What is the recommended protocol for Western blot using this antibody?

While the product information primarily recommends this antibody for ELISA and IF/ICC applications , researchers may adapt it for Western blot analysis with the following protocol considerations:

• Sample preparation:

  • Extract histones using acid extraction (0.2N HCl or 0.4N H₂SO₄)

  • Load 5-15 μg of acid-extracted histones per lane

  • Use 15% SDS-PAGE gels for optimal resolution of histone proteins

• Transfer conditions:

  • Use PVDF membrane (preferred over nitrocellulose for histone proteins)

  • Transfer at 30V overnight at 4°C for efficient transfer of small proteins

• Blocking and antibody incubation:

  • Block with 5% BSA (not milk) in TBST for 1 hour at room temperature

  • Dilute primary antibody 1:500 to 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Detection:

  • Use high-sensitivity detection reagents due to potentially low abundance of the modification

  • Include a loading control antibody against total histone H3

• Controls:

  • Include unmodified recombinant H3 as a negative control

  • If available, use synthetic 2-hydroxyisobutyrylated H3K36 peptide as a positive control

Optimization of antibody concentration and incubation conditions may be necessary.

How can I validate the specificity of this antibody for my experimental system?

Comprehensive validation of antibody specificity is essential for generating reliable scientific data. For the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody, implement the following validation approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with excess 2-hydroxyisobutyrylated H3K36 peptide

  • Compare signal with and without competing peptide

  • Specific binding should be substantially reduced or eliminated

• Modification specificity tests:

  • Compare binding to different modified peptides (acetylated K36, methylated K36, unmodified K36)

  • Use dot blot assays with peptide arrays containing various modifications

  • Specific antibody should show minimal cross-reactivity with other modifications

• Functional validation:

  • Test antibody in systems with modulated 2-hydroxyisobutyrylation levels

  • Signal should increase/decrease in accordance with biological manipulation

• Correlation with other detection methods:

  • Where possible, compare results with mass spectrometry-based detection

  • Correlation between methods supports antibody specificity

Documentation of these validation steps should accompany research findings using this antibody.

What dilution ranges should be tested during optimization?

Determining the optimal antibody concentration is critical for maximizing specific signal while minimizing background. For the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody, test the following dilution ranges for different applications:

• Immunofluorescence/ICC:

  • Start with the manufacturer's recommended range of 1:50-1:200

  • Extend testing to include 1:25 and 1:400 dilutions

  • Evaluate signal-to-noise ratio rather than absolute signal intensity

• ELISA:

  • Test dilutions from 1:100 to 1:2000

  • Generate standard curves at each dilution

  • Select dilution providing optimal detection range and sensitivity

• Western blot (if adapting):

  • Try dilutions from 1:500 to 1:2000

  • Compare signal intensity and background across the range

• ChIP (if adapting):

  • Test 1-10 μg of antibody per reaction

  • Evaluate enrichment at known positive regions vs. background

Document optimization results systematically for reproducibility and include detailed methods in publications.

How does 2-hydroxyisobutyrylation at H3K36 relate to other histone modifications?

Understanding the relationship between 2-hydroxyisobutyrylation at H3K36 and other histone modifications is crucial for deciphering the histone code. While specific studies on this relationship are still emerging, we can draw insights from related research:

H3K36 trimethylation (H3K36me3) is a well-studied modification associated with transcribed regions and deposited following RNA polymerase II elongation . This mark is recognized by "reader" proteins such as ZMYND11, which specifically binds H3K36me3 on the histone variant H3.3 and regulates transcription elongation .

Since 2-hydroxyisobutyrylation and trimethylation cannot co-exist on the same lysine residue (K36), these modifications may represent mutually exclusive marks that define different functional states or phases of transcription. The relationship may involve:

• Sequential deposition: The modifications might be placed at different stages of the transcription cycle

• Reader protein recruitment: Each modification likely recruits distinct effector proteins

• Cross-regulation: Enzymes responsible for one modification may be influenced by the presence of other modifications

• Genomic distribution patterns: ChIP-seq studies using this antibody could reveal complementary or distinct localization compared to H3K36me3

Research using the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody alongside antibodies to other modifications will help elucidate these relationships.

Can this antibody be adapted for ChIP-seq experiments?

While the manufacturer's documentation primarily specifies ELISA and IF/ICC applications , researchers interested in genome-wide mapping of 2-hydroxyisobutyrylation at H3K36 may adapt this antibody for ChIP-seq with the following considerations:

• Antibody requirements:

  • ChIP-grade antibodies typically exhibit high specificity and affinity

  • Pilot experiments should assess the antibody's performance in immunoprecipitation

• Protocol adaptations:

  • Start with 5-10 μg of antibody per ChIP reaction

  • Optimize chromatin fragmentation (aim for 200-500bp fragments)

  • Include appropriate controls (input, IgG, positive/negative genomic regions)

• Validation approaches:

  • Perform ChIP-qPCR at candidate regions before proceeding to sequencing

  • Compare enrichment patterns with known active transcription markers

  • Validate findings with biological replicates

• Data analysis considerations:

  • Use peak calling algorithms optimized for histone modifications (e.g., MACS2 with broad peak settings)

  • Compare distribution with other histone marks (e.g., H3K36me3, H3K27ac)

If adapting this antibody for ChIP-seq, thorough validation and optimization are essential for generating reliable genome-wide data.

How can I perform multiplexed detection of 2-hydroxyisobutyryl-H3K36 with other histone marks?

Multiplexed detection of histone modifications provides valuable insights into their co-occurrence and functional relationships. For detecting 2-hydroxyisobutyryl-H3K36 alongside other modifications:

• Immunofluorescence multiplexing:

  • Select antibodies raised in different host species (e.g., rabbit anti-2-hydroxyisobutyryl-H3K36 and mouse anti-H3K4me3)

  • Use species-specific secondary antibodies with non-overlapping fluorophores

  • Implement sequential staining protocols if using multiple rabbit antibodies

  • Include proper controls to account for potential cross-reactivity

• Sequential ChIP (Re-ChIP):

  • First ChIP with 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody

  • Elute bound chromatin under mild conditions

  • Perform second ChIP with antibody against another modification

  • Analyze regions containing both modifications

• Mass spectrometry approaches:

  • Perform immunoprecipitation with the antibody

  • Analyze co-occurring modifications on the same histone tail by MS/MS

  • Quantify modification combinations

Multiplexed approaches provide crucial information about the histone code complexity that cannot be obtained from single-modification studies.

What are the known biological functions of 2-hydroxyisobutyrylation at H3K36?

Based on the known role of H3K36me3 in transcription elongation and suppression of cryptic transcription , 2-hydroxyisobutyrylation at the same residue may play complementary or distinct roles in regulating gene expression. Potential functions include:

• Transcriptional regulation:

  • Possibly involved in specific phases of transcription initiation, elongation, or termination

  • May create binding sites for specific reader proteins that influence transcriptional machinery

• Chromatin organization:

  • Likely contributes to establishing active chromatin domains

  • May influence nucleosome stability or positioning

• Cell-type specific regulation:

  • Potentially involved in defining cell identity or developmental processes

  • May mark lineage-specific genes in differentiated cells

• Response to cellular conditions:

  • Could be responsive to metabolic state, as 2-hydroxyisobutyryl-CoA is a metabolic intermediate

  • May integrate metabolic signaling with gene regulation

Research using the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody will help elucidate these functions in various biological contexts.

How does 2-hydroxyisobutyrylation at H3K36 differ between cell types or conditions?

Understanding the dynamics of 2-hydroxyisobutyrylation at H3K36 across different biological contexts is an active area of research. While comprehensive studies specific to this modification are still emerging, researchers can use the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody to investigate:

• Cell type-specific patterns:

  • Compare modification levels across differentiated vs. stem cells

  • Analyze tissue-specific patterns in normal physiological contexts

  • Investigate differences between primary cells and cell lines

• Responses to cellular stimuli:

  • Measure changes following growth factor stimulation

  • Analyze modification dynamics during stress responses

  • Investigate effects of metabolic perturbations (given the link to metabolic intermediates)

• Disease-associated alterations:

  • Compare normal vs. pathological tissues or cells

  • Analyze modification changes in cancer progression

  • Investigate potential diagnostic or prognostic relevance

• Developmental dynamics:

  • Map changes during cellular differentiation

  • Analyze modification patterns during embryonic development

  • Study age-associated alterations

Systemic investigations using this antibody in various contexts will reveal the biological significance and regulatory mechanisms of this modification.

How should I quantify immunofluorescence signals for 2-hydroxyisobutyryl-H3K36?

Accurate quantification of immunofluorescence signals for histone modifications requires rigorous methodology. For the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody, consider the following quantification approach:

• Image acquisition parameters:

  • Use identical acquisition settings for all experimental and control samples

  • Ensure signals are within the dynamic range (avoid saturation)

  • Capture multiple random fields per condition (minimum 5-10)

• Nuclear signal quantification:

  • Define nuclear regions using DAPI or other nuclear counterstain

  • Measure mean fluorescence intensity within each nucleus

  • Subtract local background intensity for each measured nucleus

• Data normalization strategies:

  • Normalize to total H3 levels if performing dual staining

  • Calculate relative intensity compared to control samples

  • Consider nuclear size/DNA content if comparing different cell types

• Statistical analysis:

  • Analyze sufficient cell numbers for statistical power (typically >100 cells per condition)

  • Apply appropriate statistical tests (t-test, ANOVA) for comparing conditions

  • Report both mean values and measures of variation

• Advanced analysis:

  • Consider subnuclear distribution patterns (e.g., euchromatin vs. heterochromatin)

  • Evaluate co-localization with other nuclear markers

  • Implement machine learning approaches for pattern recognition

Free and commercial image analysis software (ImageJ, CellProfiler, etc.) can be configured for these analyses.

What genomic distribution patterns should I expect for 2-hydroxyisobutyryl-H3K36?

When analyzing ChIP-seq or similar genomic distribution data for 2-hydroxyisobutyryl-H3K36, researchers might anticipate certain patterns based on our understanding of histone modifications and the role of H3K36:

• Gene body enrichment:

  • Similar to H3K36me3 , likely shows enrichment across gene bodies of actively transcribed genes

  • May display a gradient along the gene body, possibly increasing toward the 3' end

• Relationship to gene expression:

  • Expect positive correlation with gene expression levels

  • Potentially more abundant at highly expressed genes

• Chromatin state associations:

  • Likely enriched in euchromatic regions

  • Probably depleted in heterochromatic regions and repressed genes

  • May show specific patterns at enhancers vs. promoters

• Co-occurrence patterns:

  • Possibly co-occurs with other active marks (H3K4me3, H3K27ac)

  • Mutually exclusive with H3K36me3 at the same nucleosome

  • May show specific relationship with histone variant H3.3, as observed for H3K36me3

• Cell type-specific patterns:

  • Expect differential distribution reflecting cell type-specific gene expression

  • May mark lineage-specific genes in differentiated cells

Genome-wide mapping using this antibody will help establish the precise distribution patterns of this modification.

How can I correlate 2-hydroxyisobutyryl-H3K36 patterns with gene expression?

Correlating histone modification patterns with gene expression requires integrative analysis approaches. For 2-hydroxyisobutyryl-H3K36, consider the following analytical framework:

• Data generation and preprocessing:

  • Generate matching ChIP-seq (using the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody) and RNA-seq datasets from the same samples

  • Process and normalize both datasets according to standard protocols

• Modification quantification relative to genes:

  • Calculate modification enrichment over gene bodies and/or promoter regions

  • Generate metagene profiles showing average modification distribution across genes

  • Compute per-gene enrichment scores (e.g., RPKM within gene bodies)

• Correlation analysis methods:

  • Calculate Pearson or Spearman correlation between modification enrichment and gene expression

  • Group genes by expression levels (quartiles/deciles) and compare modification profiles

  • Generate scatter plots with gene expression on one axis and modification enrichment on the other

• Advanced integrative analyses:

  • Perform time-course analysis to identify temporal relationships

  • Conduct differential analysis (correlate changes in modification with changes in expression)

  • Implement machine learning approaches to predict expression from modification patterns

• Functional interpretation:

  • Perform Gene Ontology or pathway enrichment for genes showing strong correlation

  • Compare with correlations observed for other histone marks

These analyses will help establish the functional relationship between this modification and transcriptional regulation.

What statistical approaches are appropriate for analyzing ChIP-seq data with this antibody?

• Peak identification:

  • Use peak calling algorithms optimized for broad histone modifications (MACS2 with broad settings, SICER)

  • Apply appropriate false discovery rate control (q-value < 0.05 or 0.01)

  • Consider histone-specific normalization approaches (input normalization, spike-in normalization)

• Differential binding analysis:

  • Use specialized tools (DiffBind, csaw, MAnorm) for comparative analysis between conditions

  • Apply variance modeling appropriate for ChIP-seq data

  • Control for batch effects and technical variations

• Correlation analyses:

  • Calculate genome-wide correlation with other histone marks

  • Implement hierarchical clustering to identify co-regulated regions

  • Calculate enrichment correlation with gene expression data

• Feature enrichment statistics:

  • Use permutation tests to assess enrichment at genomic features

  • Apply hypergeometric tests for overlap with other genomic annotations

  • Implement GSEA-like approaches for pathway enrichment

• Visualization and data presentation:

  • Generate browser tracks with appropriate normalization

  • Create heatmaps with statistical clustering

  • Produce metaplots with confidence intervals

How should I troubleshoot weak or non-specific signals with this antibody?

When encountering issues with signal quality using the 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody, implement this systematic troubleshooting approach:

• For weak or absent signals:

  • Increase antibody concentration (try 1:50 dilution if using 1:200)

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

  • Optimize antigen retrieval methods (mild heat-mediated retrieval)

  • Check storage conditions and antibody expiration date

  • Verify target presence in your biological system

  • Try alternative blocking reagents (5% BSA instead of serum)

• For high background or non-specific signals:

  • Increase washing steps (number and duration)

  • Decrease antibody concentration (try 1:200 if using 1:50)

  • Optimize blocking (increase time or blocker concentration)

  • Test alternative fixation protocols

  • Use more specific secondary antibodies

  • Pre-absorb the antibody with acetone powder

• For inconsistent results:

  • Standardize cell culture and experimental conditions

  • Use the same antibody lot for related experiments

  • Prepare fresh working dilutions for each experiment

  • Include positive and negative controls in each experiment

  • Implement rigorous protocol documentation

• For cross-reactivity concerns:

  • Validate with peptide competition assays

  • Compare results with other antibodies detecting the same modification

  • Perform dot blot assays with different modified peptides

Methodical documentation of optimization attempts will facilitate troubleshooting and ensure experimental reproducibility.

Future research directions for 2-hydroxyisobutyryl-H3K36 studies

The study of 2-hydroxyisobutyrylation at H3K36 represents an emerging area in epigenetic research with several promising future directions:

• Mechanistic investigations:

  • Identification of "writer" enzymes that catalyze 2-hydroxyisobutyrylation at H3K36

  • Discovery of "reader" proteins that specifically recognize this modification

  • Characterization of "eraser" enzymes that remove the modification

• Functional studies:

  • Determination of causal relationships between the modification and gene expression

  • Investigation of crosstalk with other histone modifications

  • Exploration of the role in specific biological processes (development, metabolism, stress response)

• Disease relevance:

  • Analysis of modification patterns in various pathological conditions

  • Evaluation of potential as diagnostic or prognostic biomarkers

  • Assessment as possible therapeutic targets

• Technological developments:

  • Generation of more specific antibodies and detection tools

  • Development of site-specific approaches to manipulate the modification

  • Integration with emerging single-cell epigenomic technologies

The 2-hydroxyisobutyryl-HIST1H3A (K36) Antibody will be a valuable tool in advancing these research directions by enabling specific detection and mapping of this modification.