2-hydroxyisobutyryl-HIST1H3A (K18) Antibody

What is the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody and what does it detect?

The 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody is a primary polyclonal antibody that specifically recognizes and binds to the 2-hydroxyisobutyryl modification at lysine 18 of Histone H3.1 (HIST1H3A). This antibody enables researchers to study this specific post-translational modification in the context of epigenetic regulation .

Histone H3.1 is a core component of nucleosomes that wrap and compact DNA into chromatin, thereby limiting DNA accessibility to cellular machinery. This protein plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability .

What are the key specifications of the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody?

The 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody has the following specifications:

These specifications are crucial for experimental planning and execution .

What applications is this antibody validated for?

The 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody has been validated for multiple experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Recommended dilution 1:2000-1:10000

• WB (Western Blot): Recommended dilution 1:500-1:2000

• ICC (Immunocytochemistry): Recommended dilution 1:20-1:200

• IF (Immunofluorescence): Recommended dilution 1:20-1:200

• ChIP (Chromatin Immunoprecipitation)

This versatility makes the antibody suitable for diverse experimental approaches in epigenetic research .

How should I design Western Blot experiments using this antibody?

For optimal Western Blot results with the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody:

• Sample preparation: Use whole cell lysates from human cell lines such as HeLa, Jurkat, HEK293, or HepG2.

• Treatment considerations: Consider treating cells with 30mM sodium butyrate for 4 hours to enhance the histone modification signal.

• Loading controls: Include appropriate loading controls for histone proteins.

• Antibody dilution: Use a 1:500 dilution of the primary antibody.

• Secondary antibody: Apply a goat polyclonal to rabbit IgG at approximately 1:40000 dilution.

• Expected results: Look for a band at approximately 16 kDa, which is the predicted molecular weight for Histone H3.1.

This methodology has been validated with multiple human cell lines and demonstrates consistent results across different cell types .

What is the recommended protocol for Immunofluorescence staining?

For immunofluorescence applications:

• Cell preparation: Culture cells on appropriate coverslips or slides.

• Treatment: For enhanced signal, consider treating cells with 30mM sodium butyrate for 4 hours.

• Fixation: Fix cells in 4% formaldehyde.

• Permeabilization: Permeabilize using 0.2% Triton X-100.

• Blocking: Block in 10% normal Goat Serum.

• Primary antibody incubation: Incubate with the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody at a 1:50 dilution overnight at 4°C.

• Secondary antibody: Use Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).

• Counterstain: DAPI is recommended for nuclear visualization.

This protocol has been successfully used with HeLa cells and should provide clear nuclear staining patterns that correspond to the localization of the modified histone .

How can I implement this antibody in Immunocytochemistry experiments?

For Immunocytochemistry applications:

• Cell preparation: Culture cells on appropriate slides or plates.

• Treatment: Consider treating cells with 30mM sodium butyrate for 4 hours to enhance modification levels.

• Fixation: Fix cells in 4% formaldehyde.

• Permeabilization: Use 0.2% Triton X-100.

• Blocking: Block with 10% normal goat serum for 30 minutes at room temperature.

• Primary antibody: Dilute the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody at 1:100 in 1% BSA and incubate overnight at 4°C.

• Detection system: Use a biotinylated secondary antibody and visualize using an HRP conjugated SP system.

This method has been validated with HeLa cells and provides specific nuclear staining patterns .

How does the 2-hydroxyisobutyryl modification at K18 differ from other histone post-translational modifications?

The 2-hydroxyisobutyrylation of histone H3 at lysine 18 (K18) represents a distinct post-translational modification that differs from better-known modifications such as acetylation, methylation, or phosphorylation. This modification involves the addition of a 2-hydroxyisobutyryl group specifically to the lysine residue at position 18 of the histone H3.1 protein.

Key distinguishing features:

• Chemical structure: The 2-hydroxyisobutyryl group is bulkier than acetyl groups, potentially causing more significant structural changes to chromatin.

• Regulatory functions: While acetylation typically correlates with gene activation, 2-hydroxyisobutyrylation may have distinct regulatory roles in chromatin dynamics and gene expression.

• Enzymatic regulation: The enzymes responsible for adding and removing this modification likely differ from histone acetyltransferases (HATs) and histone deacetylases (HDACs).

• Cellular context: Studies using sodium butyrate treatment (30mM for 4h) suggest that this modification may be regulated by metabolic factors or stress conditions .

What are the best experimental approaches to distinguish between 2-hydroxyisobutyrylation at K18 versus other histone modifications?

To distinguish between 2-hydroxyisobutyrylation at K18 and other histone modifications:

• Antibody specificity validation:

  • Perform peptide competition assays using modified and unmodified peptides

  • Conduct dot blot analysis with various modified histone peptides

  • Use multiple antibodies targeting different epitopes to confirm specificity

• Mass spectrometry approaches:

  • Implement tandem mass spectrometry (MS/MS) to definitively identify the modification

  • Use high-resolution mass spectrometry to distinguish between isobaric modifications

  • Consider targeted methods such as Multiple Reaction Monitoring (MRM) for quantification

• Combinatorial analysis:

  • Investigate co-occurrence with other histone modifications through sequential immunoprecipitation

  • Perform ChIP-seq experiments to map genomic distribution

  • Compare with datasets for other histone modifications to identify unique patterns

• Functional studies:

  • Employ site-directed mutagenesis of K18 to evaluate functional consequences

  • Utilize CRISPR-Cas9 to target enzymes responsible for the modification

  • Combine with gene expression analysis to determine regulatory impacts

How can ChIP experiments be optimized for the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody?

For optimal Chromatin Immunoprecipitation (ChIP) using the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.75-1.5%)

  • Optimize crosslinking times (10-15 minutes) for best results

  • Consider dual crosslinking with formaldehyde and protein-specific crosslinkers

• Chromatin preparation:

  • Sonicate chromatin to achieve fragments of 200-500 bp

  • Verify fragmentation through agarose gel electrophoresis

  • Pre-clear chromatin with protein A/G beads to reduce background

• Immunoprecipitation conditions:

  • Use 2-5 μg of antibody per ChIP reaction

  • Incubate overnight at 4°C with gentle rotation

  • Include appropriate controls (IgG control, input samples)

• Washing and elution:

  • Implement stringent washing steps to reduce non-specific binding

  • Consider sequential ChIP for co-occurrence studies with other modifications

  • Optimize elution conditions for maximum recovery

• Downstream analysis:

  • Perform qPCR on known target regions

  • Consider genome-wide approaches like ChIP-seq

  • Analyze data with appropriate normalization to input and controls

What are the expected results and potential pitfalls when using this antibody in Western Blot applications?

Expected results and troubleshooting for Western Blot applications:

The 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody typically produces a clear band at 16 kDa in Western blot analysis of human cell lysates. The signal is often enhanced when cells are treated with sodium butyrate, which may increase the levels of this modification .

How can researchers validate the specificity of the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody?

To validate antibody specificity:

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide (modified at K18)

  • Compare with control (no peptide) and non-specific peptide competitions

  • A specific antibody will show significantly reduced signal when pre-incubated with the specific peptide

• Knockout/knockdown validation:

  • Use genetic approaches to reduce histone H3.1 expression

  • Alternatively, mutate K18 to a non-modifiable residue

  • Confirm reduction/elimination of the signal

• Cross-reactivity testing:

  • Test against other histone 2-hydroxyisobutyrylation sites (e.g., K27, K56)

  • Use peptide arrays with various histone modifications

  • Perform dot blots with differentially modified recombinant histones

• Correlation with other detection methods:

  • Compare results with mass spectrometry data

  • Use alternative antibodies targeting the same modification

  • Verify localization patterns match expected nuclear distribution

What controls should be included in experiments using this antibody?

Essential controls for experiments using the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody:

• Positive controls:

  • Lysates from cells treated with 30mM sodium butyrate for 4h

  • Recombinant histone H3.1 with 2-hydroxyisobutyrylation at K18

  • Previously validated positive samples

• Negative controls:

  • Isotype control (rabbit IgG) at equivalent concentration

  • Untreated cell samples (for comparison with sodium butyrate treatment)

  • Peptide competition control

• Loading/technical controls:

  • Total histone H3 antibody to normalize for histone content

  • GAPDH or β-actin for total protein loading (in Western blots)

  • Nuclear staining (DAPI) for localization studies

• Validation controls:

  • Modified peptide dot blots

  • Sequential dilutions to verify signal linearity

  • Multiple cell lines to verify consistency of results

• Treatment controls:

  • HDAC inhibitors other than sodium butyrate

  • Time course of treatment to establish optimal conditions

  • Dose-response experiments to determine sensitivity

How can the 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody be used to study epigenetic regulation in disease models?

The 2-hydroxyisobutyryl-HIST1H3A (K18) Antibody enables researchers to investigate this histone modification in various disease contexts:

• Cancer research applications:

  • Compare 2-hydroxyisobutyrylation levels between normal and tumor tissues

  • Correlate modification patterns with gene expression changes in oncogenesis

  • Investigate whether altered metabolism in cancer cells affects this modification

  • Study potential cooperation with other epigenetic marks in cancer progression

• Neurodegenerative disease models:

  • Examine age-dependent changes in 2-hydroxyisobutyrylation patterns

  • Investigate interactions with disease-associated proteins

  • Correlate with altered gene expression in affected brain regions

  • Study potential therapeutic interventions targeting this modification

• Metabolic disorders:

  • Investigate how metabolic stress affects 2-hydroxyisobutyrylation levels

  • Study the relationship between cellular metabolism and this modification

  • Examine tissue-specific patterns in metabolic disease models

  • Correlate with transcriptional changes in metabolic pathways

• Experimental methodologies:

  • ChIP-seq to map genome-wide modification patterns in disease states

  • Integrate with RNA-seq data to correlate with transcriptional changes

  • Combine with other epigenetic marks to build comprehensive regulatory maps

  • Develop targeted therapeutic approaches based on identified patterns

What is the relationship between 2-hydroxyisobutyrylation at K18 and other histone H3 modifications?

The relationship between 2-hydroxyisobutyrylation at K18 and other histone H3 modifications is complex and represents an important area of epigenetic research:

• Modification crosstalk:

  • 2-hydroxyisobutyrylation may compete with acetylation at K18

  • Adjacent modifications (e.g., K14ac, K23ac) may influence K18 2-hydroxyisobutyrylation

  • H3S10 phosphorylation could affect recognition or establishment of K18 2-hydroxyisobutyrylation

  • H3K9 or H3K27 methylation status may correlate with K18 2-hydroxyisobutyrylation patterns

• Temporal dynamics:

  • Sequential modification patterns during cell cycle progression

  • Changes during cellular differentiation or response to stimuli

  • Establishment and maintenance during DNA replication

• Functional consequences:

  • Effects on chromatin structure compared to other modifications

  • Differential recruitment of reader proteins

  • Impact on transcriptional machinery access

  • Role in specialized processes like DNA repair or replication

• Experimental approaches to study relationships:

  • Sequential ChIP (re-ChIP) to identify co-occurrence

  • Mass spectrometry of histone modification combinations

  • Super-resolution microscopy to visualize spatial relationships

  • Genetic approaches to manipulate specific modifications and observe effects on others

How can researchers integrate 2-hydroxyisobutyryl-HIST1H3A (K18) ChIP-seq data with other genomic datasets?

For integrative analysis of 2-hydroxyisobutyryl-HIST1H3A (K18) ChIP-seq data:

• Multi-omics integration strategies:

  • Correlate with RNA-seq data to identify transcriptional impacts

  • Integrate with DNA methylation profiles to understand epigenetic interactions

  • Combine with ATAC-seq or DNase-seq to assess chromatin accessibility

  • Compare with other histone modification ChIP-seq datasets

• Computational analysis approaches:

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

  • Perform differential binding analysis between experimental conditions

  • Use genome segmentation tools (e.g., ChromHMM) to identify chromatin states

  • Apply machine learning approaches to identify combinatorial patterns

• Visualization and interpretation:

  • Generate heat maps centered on transcription start sites or enhancers

  • Create genome browser tracks to visualize distribution patterns

  • Perform motif analysis to identify associated transcription factors

  • Conduct pathway enrichment analysis for genes associated with this modification

• Functional validation approaches:

  • Integrate with HiC or chromatin conformation capture data

  • Correlate with transcription factor binding patterns

  • Validate key findings with targeted experiments (luciferase assays, gene expression)

  • Use CRISPR-based epigenome editing to manipulate specific sites

What are emerging trends in research using 2-hydroxyisobutyryl-HIST1H3A antibodies?

Emerging research trends with 2-hydroxyisobutyryl-HIST1H3A antibodies include:

• Single-cell applications:

  • Adaptation of techniques for single-cell ChIP-seq

  • Integration with single-cell RNA-seq for correlation studies

  • Development of high-sensitivity detection methods for limited samples

• In vivo dynamics:

  • Live-cell imaging using labeled antibody fragments

  • Development of modification-specific biosensors

  • Real-time monitoring of modification changes during cellular processes

• Therapeutic targeting:

  • Identification of enzymes regulating this modification

  • Development of small molecules targeting these enzymes

  • Potential for epigenetic therapy approaches

• Cross-species comparative studies:

  • Evolution of this modification across species

  • Conservation of regulatory mechanisms

  • Functional divergence in different organisms

The continued development of highly specific antibodies against various histone modifications, including 2-hydroxyisobutyrylation, will enable more sophisticated understanding of the histone code and its biological significance .

What methodological advances are needed to better study 2-hydroxyisobutyryl-HIST1H3A (K18) in complex biological systems?

Future methodological advances needed for studying 2-hydroxyisobutyryl-HIST1H3A (K18):

• Technical improvements:

  • Development of homogeneous antibody preparations with defined epitope recognition

  • Creation of recombinant antibody formats for improved reproducibility

  • Generation of modification-specific nanobodies for advanced applications

  • Improved methods for fixed tissue ChIP applications

• Multi-modification analysis:

  • Development of multiplexed ChIP approaches for simultaneous detection

  • Improved mass spectrometry methods for quantitative analysis of modification combinations

  • Advanced imaging techniques for visualization of modification co-occurrence

• Functional analysis tools:

  • CRISPR-based tools for site-specific modification introduction or removal

  • Development of selective readers or erasers for this modification

  • High-throughput screening systems to identify regulatory factors

• Computational approaches:

  • Advanced algorithms for integration of multi-omics data

  • Predictive modeling of modification dynamics

  • Machine learning approaches to identify subtle modification patterns