2-hydroxyisobutyryl-HIST1H3A (K27) Antibody

What is 2-hydroxyisobutyryl-HIST1H3A (K27) and what cellular functions does it regulate?

2-hydroxyisobutyryl-HIST1H3A (K27) refers to histone H3.1 that has been modified with a 2-hydroxyisobutyryl group at the lysine 27 position. Histone H3 is a core component of nucleosomes, which wrap and compact DNA into chromatin. This compaction limits DNA accessibility to cellular machinery that requires DNA as a template. Histone modifications, including 2-hydroxyisobutyrylation, are part of the "histone code" that regulates DNA accessibility .

While specific functions of 2-hydroxyisobutyrylation at K27 are still being elucidated, it likely plays roles similar to other histone modifications in transcription regulation, DNA repair, DNA replication, and chromosomal stability. Based on patterns observed with other modifications, it may mark specific chromatin states that either promote or repress gene expression depending on the genomic context and co-occurring modifications .

Unlike the better-characterized H3K27 acetylation (H3K27ac), which is known to mark active enhancers and promoters, the 2-hydroxyisobutyryl modification may have distinct functions that warrant further investigation using specific antibodies designed to recognize this particular modification .

How does the 2-hydroxyisobutyryl-HIST1H3A (K27) antibody specificity compare to other H3K27 modification antibodies?

Antibody specificity is critical when studying histone modifications, as closely related modifications can be difficult to distinguish. The 2-hydroxyisobutyryl-HIST1H3A (K27) polyclonal antibody is specifically designed to recognize the 2-hydroxyisobutyryl modification at lysine 27 of histone H3.1 .

Research on other histone modification antibodies has shown that antibody binding can be affected by neighboring modifications. For example, phosphorylation of serine residues adjacent to lysine methylation sites (such as S28 next to K27) can interfere with antibody recognition, as observed with H3K27 methylation-specific antibodies . Similarly, the 2-hydroxyisobutyryl-HIST1H3A (K27) antibody specificity could potentially be affected by modifications at neighboring residues, such as R26 methylation or citrullination, or S28 phosphorylation.

When selecting an antibody for your research, it's important to verify that the antibody has been validated for specificity against the target modification and lacks cross-reactivity with other similar modifications. Validation methods should include peptide competition assays and tests with samples containing known modifications .

What are the recommended applications for 2-hydroxyisobutyryl-HIST1H3A (K27) antibody?

The 2-hydroxyisobutyryl-HIST1H3A (K27) polyclonal antibody has been validated for several applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of the modification in purified histones or nuclear extracts

• WB (Western Blot): For detecting the modification in protein samples separated by gel electrophoresis

• ICC (Immunocytochemistry): For visualizing the modification in fixed cells

• IF (Immunofluorescence): For fluorescent visualization of the modification in cells or tissues

Based on similar antibodies like the H3K27ac antibody, additional applications might include:

• ChIP (Chromatin Immunoprecipitation): For identifying genomic regions enriched for this modification

• Flow cytometry: For quantifying the modification in individual cells

For optimal results in each application, follow the manufacturer's recommended protocols for antibody concentration, incubation conditions, and detection methods. Research has shown that antibody concentration may not be crucial for obtaining quantitative immunofluorescence data, as similar modification profiles have been observed across different antibody concentrations (0.25-4 μg/ml) .

What controls should be included when using the 2-hydroxyisobutyryl-HIST1H3A (K27) antibody?

When working with histone modification antibodies, appropriate controls are essential to ensure data reliability:

• Peptide competition assay: Pre-incubate the antibody with excess modified peptide (2-hydroxyisobutyryl-K27) to confirm specificity. Signal should be significantly reduced or eliminated.

• Positive control samples: Include samples known to contain the 2-hydroxyisobutyryl-K27 modification. For cell-based experiments, consider cell types or treatments that enhance this modification.

• Negative controls:

  • Unmodified peptide or histone

  • Samples treated with inhibitors of enzymes that catalyze 2-hydroxyisobutyrylation

  • Secondary antibody-only controls to assess background signal

• Cross-reactivity controls: Test the antibody against peptides with similar modifications (e.g., acetylation at K27) to ensure specificity .

• Technical validation: When performing multicolor immunofluorescence, validate that one antibody does not interfere with the binding of another through steric hindrance. Research has shown that combinations of antibodies recognizing modifications on the same histone (such as H3K27un and H3K36me3) can be used without interference when antibody binding is not at saturation levels .

How does 2-hydroxyisobutyryl-HIST1H3A (K27) level change during the cell cycle?

While specific data for 2-hydroxyisobutyryl-HIST1H3A (K27) dynamics throughout the cell cycle is limited in the provided search results, we can draw parallels from studies of other histone modifications.

Research on histone modifications during the cell cycle has revealed distinct patterns. Active marks, including acetylation of various residues, tend to increase during S phase in association with chromatin duplication . For example, H4K5ac increases significantly during S phase, reflecting the acetylation of newly assembled histones .

If 2-hydroxyisobutyryl-K27 functions as an active mark similar to acetylation, it might follow a similar pattern with:

• Increased levels during S phase when chromatin is duplicated

• Possible reduction during G2 phase as repressive marks are established

• Restoration dynamics that may occur within the same cell cycle or extend to the next G1 phase

To investigate this question experimentally:

• Synchronize cells at different cell cycle stages using methods such as double thymidine block or nocodazole treatment

• Perform immunofluorescence with the 2-hydroxyisobutyryl-HIST1H3A (K27) antibody

• Co-stain with markers for cell cycle phases (e.g., H4K5ac for S phase)

• Quantify modification levels using image analysis software

• Calculate correlations between the 2-hydroxyisobutyryl-K27 signal and cell cycle markers

This approach would allow mapping of the dynamic changes in this modification throughout the cell cycle, providing insights into its regulatory mechanisms and functions.

What is the relationship between 2-hydroxyisobutyryl-HIST1H3A (K27) and other histone modifications?

Understanding the relationship between different histone modifications is crucial for deciphering the histone code. While specific data on 2-hydroxyisobutyryl-K27 co-occurrence with other modifications is not detailed in the search results, we can outline a methodological approach to investigate these relationships:

• Multicolor immunofluorescence analysis: This technique allows simultaneous detection of multiple histone modifications in single cells . By directly labeling modification-specific antibodies with different fluorophores, you can visualize and quantify up to four histone modifications simultaneously .

• Correlation analysis: Calculate Pearson correlation coefficients between 2-hydroxyisobutyryl-K27 and other modifications across many cells. This approach has been used to classify histone modifications into groups based on their cell cycle dynamics .

• Sequential ChIP (Re-ChIP): Perform ChIP first with 2-hydroxyisobutyryl-K27 antibody, then with antibodies against other modifications to identify genomic regions containing both modifications.

Based on findings with other modifications, 2-hydroxyisobutyryl-K27 might show positive correlations with active marks (like H3K4me3 and acetylation marks) if it promotes gene expression, or with repressive marks (like H3K9me3 or H3K27me3) if it is associated with gene silencing .

When designing co-staining experiments, consider that antibody combinations recognizing modifications that are relatively close and co-exist on a single histone molecule (such as H3K4un and H3K9me2, or H3K27un and H3K36me3) have been successfully used without interference under non-saturating antibody concentrations .

How can ChIP-seq be optimized for 2-hydroxyisobutyryl-HIST1H3A (K27) antibodies?

Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique for genome-wide mapping of histone modifications. Optimizing ChIP-seq for 2-hydroxyisobutyryl-HIST1H3A (K27) antibodies requires careful consideration of several parameters:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (typically 0.75-1%) and incubation times (8-15 minutes)

  • Consider dual crosslinking with additional agents like EGS (ethylene glycol bis-succinimidyl succinate) for improved efficiency

• Sonication parameters:

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

  • Verify fragment size by agarose gel electrophoresis before proceeding

• Antibody titration:

  • Determine the optimal antibody amount through titration experiments

  • For polyclonal antibodies like the 2-hydroxyisobutyryl-HIST1H3A (K27) antibody, typically 2-5 μg per ChIP reaction is a good starting point

• IP conditions:

  • Optimize incubation time and temperature (typically overnight at 4°C)

  • Consider pre-clearing chromatin with protein A/G beads to reduce background

• Controls:

  • Include input chromatin (non-immunoprecipitated) control

  • Include IgG negative control

  • Consider including a spike-in normalization control

• Validation of ChIP efficiency:

  • Before sequencing, validate enrichment at known or expected sites by qPCR

  • Calculate percent input or fold enrichment over IgG control

• Sequencing considerations:

  • Aim for at least 20 million uniquely mapped reads per sample

  • Consider paired-end sequencing for improved mapping accuracy

• Bioinformatic analysis:

  • Use appropriate peak calling algorithms (e.g., MACS2)

  • Apply normalization methods suitable for histone modification data

For analyzing the relationship between 2-hydroxyisobutyryl-K27 and gene expression, integrate ChIP-seq data with RNA-seq data from the same cell type or condition to identify correlations between modification enrichment and transcriptional activity.

What methodological considerations are important for multiplexed analysis of 2-hydroxyisobutyryl alongside other histone modifications?

Multiplexed analysis of histone modifications provides valuable insights into the combinatorial nature of the histone code. When including 2-hydroxyisobutyryl-HIST1H3A (K27) in multiplexed analyses, consider the following methodological aspects:

• Antibody compatibility and specificity:

  • Select antibodies raised in different host species to allow simultaneous detection

  • If using antibodies from the same species, consider direct fluorophore labeling to avoid cross-reactivity

  • Validate that antibody combinations do not interfere with each other's binding through steric hindrance

• Optimizing immunofluorescence protocols:

  • Antibody concentration: Research has shown that antibody profiles remain consistent across a range of concentrations (0.25-4 μg/ml), suggesting that exact concentration is not crucial for obtaining quantitative data

  • Sequential vs. simultaneous staining: Test both approaches to determine which provides the best signal-to-noise ratio for your specific antibody combination

• Image acquisition and analysis:

  • Use a microscope with appropriate filter sets to avoid bleed-through between fluorescent channels

  • Collect sufficient numbers of cells (400-500 nuclei) for robust statistical analysis

  • Implement automated image analysis for quantification of nuclear signal intensities

• Data analysis for co-occurrence patterns:

  • Use correlation analysis to identify relationships between modifications

  • Apply clustering algorithms to group cells with similar modification patterns

  • Consider dimensionality reduction techniques (PCA, t-SNE) for visualizing complex relationships

• Cell cycle considerations:

  • Co-stain with cell cycle markers (e.g., H4K5ac for S phase) to account for cell cycle-dependent variation

  • Analyze modification correlations within specific cell cycle phases to avoid confounding effects

A multicolor immunofluorescence approach, as described in reference , provides a powerful method for analyzing multiple histone modifications in single cells. This approach has been successfully applied to study histone modification dynamics during the cell cycle and could be adapted to include 2-hydroxyisobutyryl-K27 analysis.

What are common issues when working with 2-hydroxyisobutyryl-HIST1H3A (K27) antibodies and how can they be resolved?

When working with histone modification antibodies, including those targeting 2-hydroxyisobutyryl-HIST1H3A (K27), researchers may encounter several technical challenges. Here are common issues and their solutions:

Research on other histone modification antibodies has shown that antibody binding can be affected by modifications at adjacent residues. For example, H3K27me-specific antibodies cannot bind when the neighboring S28 is phosphorylated . Similarly, 2-hydroxyisobutyryl-K27 antibody binding might be affected by modifications at neighboring residues like R26 or S28.

To verify antibody specificity, perform peptide competition assays using peptides containing the 2-hydroxyisobutyryl-K27 modification alone or in combination with modifications at adjacent residues. This will help determine whether the antibody's binding is affected by neighboring modifications.

How can sample preparation be optimized for detecting 2-hydroxyisobutyryl-HIST1H3A (K27)?

Sample preparation is crucial for reliable detection of histone modifications. For optimal detection of 2-hydroxyisobutyryl-HIST1H3A (K27), consider the following protocol optimizations:

• Cell/tissue fixation:

  • For immunofluorescence and immunohistochemistry: Fix samples with 4% paraformaldehyde for 10-15 minutes at room temperature

  • Avoid overfixation, which can mask epitopes

  • For certain applications, methanol fixation may provide better epitope accessibility

• Permeabilization:

  • Use 0.1-0.5% Triton X-100 for 5-10 minutes to allow antibody access to nuclear antigens

  • For delicate samples, consider milder detergents like 0.1% Saponin

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) can improve detection

  • Test different retrieval methods to determine optimal conditions for the 2-hydroxyisobutyryl-K27 antibody

• Blocking:

  • Use 3-5% BSA or normal serum (from the species of the secondary antibody) in PBS

  • Include 0.1% Triton X-100 in blocking solution to reduce background

• Antibody incubation:

  • For immunofluorescence, concentrations between 0.25-4 μg/ml have been shown to provide consistent results

  • Incubate primary antibody overnight at 4°C for optimal binding

  • For multicolor immunofluorescence, directly labeled antibodies can be used simultaneously without interference when not at saturating concentrations

• Protein extraction for Western blot:

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

  • Include histone deacetylase inhibitors (e.g., sodium butyrate) and protease inhibitors in extraction buffers

  • Consider adding 2-hydroxyisobutyryl modification inhibitors if these become identified

• ChIP sample preparation:

  • Crosslink chromatin with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine

  • Optimize sonication conditions to generate 200-500bp fragments

Proper sample preparation can significantly impact the sensitivity and specificity of 2-hydroxyisobutyryl-HIST1H3A (K27) detection, leading to more reliable and reproducible results.