2-hydroxyisobutyryl-HIST1H3A (K122) Antibody

What is 2-hydroxyisobutyrylation of histone H3 and why is it significant in epigenetics research?

2-hydroxyisobutyrylation is a post-translational modification (PTM) of histones that plays a crucial role in epigenetic regulation. This modification occurs at specific lysine residues on histone proteins, including H3, and contributes to chromatin structure modulation and gene expression regulation. In the context of HIST1H3A, 2-hydroxyisobutyrylation at lysine positions (such as K18 or K122) represents an important epigenetic mark that researchers study to understand chromatin dynamics and transcriptional control mechanisms. This modification is part of the expanding "histone code" that determines genome function beyond the DNA sequence itself .

How do 2-hydroxyisobutyryl-HIST1H3A antibodies differ based on their target lysine residues (K18 vs. K122)?

The key difference between these antibodies is their target epitope specificity - they recognize 2-hydroxyisobutyryl modifications at distinct lysine positions on the histone H3.1 protein. The K18 antibody targets the modification at lysine 18, while the K122 antibody specifically recognizes the modification at lysine 122. These different lysine positions are located in distinct regions of the histone protein and likely serve different functional roles in chromatin regulation. The immunogens used to develop these antibodies contain peptide sequences surrounding the specifically modified lysine residue (K18 or K122) derived from human histone H3.1 . Researchers should select the appropriate antibody based on which specific modification site they need to investigate for their particular epigenetic research question.

What are the validated applications for 2-hydroxyisobutyryl-HIST1H3A antibodies, and which techniques yield optimal results?

2-hydroxyisobutyryl-HIST1H3A antibodies have been validated for multiple experimental applications:

For optimal results across these applications, researchers should perform careful antibody titration experiments to determine the ideal concentration for their specific sample type and experimental conditions. Western blotting typically yields excellent quantitative results for measuring global changes in histone modifications, while ChIP provides insights into genomic localization of the modifications .

What is the recommended protocol for monitoring 2-hydroxyisobutyryl-HIST1H3A modifications using flow cytometry?

Based on protocols established for histone modification analysis, a flow cytometry approach can be adapted for 2-hydroxyisobutyryl-HIST1H3A detection. The method involves:

• Cell fixation with formaldehyde (typically 1-4%) for 10-15 minutes at room temperature

• Permeabilization with ice-cold 90% methanol for 30 minutes

• Blocking with PBS containing 0.5% BSA for 10 minutes

• Incubation with primary 2-hydroxyisobutyryl-HIST1H3A antibody (1:50-1:200 dilution) for 1 hour

• Washing with PBS containing 0.5% BSA (3×)

• Incubation with fluorophore-conjugated secondary antibody for 30 minutes

• Final washing and analysis by flow cytometry

Flow cytometry offers advantages for quantitative assessment of histone modifications in individual cells and can detect relative differences in modification levels with high sensitivity. Studies have shown that flow cytometry may provide superior quantitative results compared to western blotting for monitoring histone modifications in certain contexts .

How should samples be prepared and stored to preserve 2-hydroxyisobutyryl modifications for antibody detection?

To maintain the integrity of 2-hydroxyisobutyryl modifications for reliable antibody detection:

• Sample Collection: Harvest cells or tissues rapidly and process immediately to prevent loss of modifications

• Fixation: Use freshly prepared fixatives (4% paraformaldehyde or formaldehyde) when applicable

• Protein Extraction: Include HDAC inhibitors (e.g., sodium butyrate) and protease inhibitors in lysis buffers

• Storage Conditions:

  • Short-term (≤1 week): 4°C in appropriate buffer with preservatives

  • Long-term: -20°C or -80°C with 50% glycerol and protective agents (0.03% Proclin 300, 0.01M PBS, pH 7.4)

  • Avoid repeated freeze-thaw cycles

Research has shown that histone modifications can be reliably detected for up to 96 hours at various temperatures (4°C, 20°C, or 37°C), though stability decreases significantly after 96 hours at 37°C. For optimal results, analyze samples as soon as possible after collection, especially when quantitative comparisons are critical .

What are the critical considerations for lysine 122 modification preservation compared to other histone modification sites?

The preservation of lysine 122 modifications requires specific attention due to its location in the C-terminal region of histone H3, which may affect accessibility and stability differently than modifications at other sites like K18:

• Extraction Methods: Use specialized histone extraction protocols that maintain the integrity of the histone C-terminal domain

• Buffer Composition: Include deacetylase inhibitors specific to the enzymes that target K122 modifications

• Sample Processing Time: Minimize processing time to prevent enzymatic removal of the modifications

• Denaturing Conditions: Adjust denaturing conditions during western blotting procedures to ensure proper epitope exposure

• Fixation Parameters: For immunofluorescence or ChIP applications, optimize fixation parameters specifically for K122 accessibility

These considerations are particularly important since the dynamics and stability of different histone modifications can vary based on their position within the histone protein and their functional roles in chromatin regulation .

How can 2-hydroxyisobutyryl-HIST1H3A antibodies be used to investigate the crosstalk between different histone modifications?

To investigate modification crosstalk using 2-hydroxyisobutyryl-HIST1H3A antibodies:

• Sequential ChIP (Re-ChIP): Perform initial ChIP with 2-hydroxyisobutyryl-HIST1H3A antibody, followed by a second immunoprecipitation with antibodies against other modifications to identify co-occurrence patterns

• Mass Spectrometry Integration: Combine antibody-based enrichment with mass spectrometry to identify and quantify multiple modifications on the same histone molecules

• Proximity Ligation Assays: Detect spatial proximity between 2-hydroxyisobutyryl modifications and other histone marks within the same nucleosome

• Combinatorial Immunofluorescence: Use multiplexed immunofluorescence with antibodies against different modifications to assess correlation patterns

• Genome-wide ChIP-seq Analysis: Compare ChIP-seq profiles of 2-hydroxyisobutyryl-HIST1H3A with other histone marks to identify regions of overlap or mutual exclusivity

This integrated approach can reveal functional relationships between 2-hydroxyisobutyryl modifications and other epigenetic marks such as acetylation, methylation, or phosphorylation, providing insights into the complex regulatory mechanisms of the histone code .

What are the optimal strategies for multiplexing 2-hydroxyisobutyryl-HIST1H3A antibodies with other histone modification antibodies?

For effective multiplexing of 2-hydroxyisobutyryl-HIST1H3A antibodies with other histone modification antibodies:

• Antibody Selection Criteria:

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

  • Verify absence of cross-reactivity between antibodies

  • Select antibodies with compatible working dilutions

• Fluorophore Combination Strategies:

  • Use fluorophores with minimal spectral overlap

  • Apply appropriate compensation controls

  • Consider brightness hierarchy based on expected target abundance

• Sequential Staining Protocols:

  • Block between sequential antibody applications

  • Consider mild stripping between applications if necessary

  • Optimize order of antibody application to minimize interference

• Validation Approaches:

  • Perform single-antibody controls alongside multiplexed experiments

  • Include biological controls with known modification patterns

  • Validate results using alternative techniques

By carefully implementing these strategies, researchers can simultaneously analyze multiple histone modifications, enabling more comprehensive insights into the complex relationships within the epigenetic landscape .

What are common sources of non-specific binding with 2-hydroxyisobutyryl-HIST1H3A antibodies and how can they be mitigated?

Common sources of non-specific binding and their mitigation strategies include:

Additionally, researchers should incorporate proper controls including:

• No primary antibody control

• Peptide competition assays with modified and unmodified peptides

• Samples with known modification status (positive and negative controls)

These measures help ensure that the observed signals are specific to the 2-hydroxyisobutyryl modification at the intended lysine position (K122 or K18) .

How can researchers validate the specificity of 2-hydroxyisobutyryl-HIST1H3A antibodies for their particular experimental system?

To validate antibody specificity for 2-hydroxyisobutyryl-HIST1H3A in a specific experimental system:

• Peptide Competition Assays:

  • Pre-incubate antibody with excess modified peptide (containing 2-hydroxyisobutyryl-K122 or K18)

  • Pre-incubate with unmodified peptide as control

  • Observe signal reduction only with the modified peptide

• Modification Enzyme Manipulation:

  • Treat samples with HDAC inhibitors to increase modification levels

  • Compare signal intensity before and after treatment

  • Observe expected changes in signal intensity

• Knockdown/Knockout Validation:

  • Deplete enzymes responsible for 2-hydroxyisobutyryl modification

  • Confirm reduction in signal using the antibody

• Mass Spectrometry Correlation:

  • Analyze samples by both antibody-based detection and mass spectrometry

  • Confirm correlation between methods for modification abundance

• Signal Localization Analysis:

  • Verify nuclear localization consistent with histone proteins

  • Confirm expected pattern distribution in chromatin

This comprehensive validation ensures that the antibody specifically recognizes the 2-hydroxyisobutyryl modification at the correct lysine residue in the particular experimental context .

How does detection of 2-hydroxyisobutyryl-HIST1H3A compare methodologically with detection of other histone H3 modifications?

Methodological comparison between 2-hydroxyisobutyryl-HIST1H3A and other histone H3 modifications:

For optimal results across modification types, flow cytometry has proven particularly effective for quantitative analysis, offering superior sensitivity compared to western blotting in some contexts. Research has demonstrated that histone H3 modifications can be reliably detected across various storage conditions, though stability decreases significantly after prolonged storage at higher temperatures .

What are the emerging applications of 2-hydroxyisobutyryl-HIST1H3A antibodies in integrated multi-omics research approaches?

Emerging applications of 2-hydroxyisobutyryl-HIST1H3A antibodies in multi-omics research include:

• Epigenome-Transcriptome Integration:

  • ChIP-seq with 2-hydroxyisobutyryl-HIST1H3A antibodies combined with RNA-seq

  • Correlation of 2-hydroxyisobutyryl modification patterns with gene expression profiles

  • Identification of regulatory elements marked by this modification

• Chromatin Accessibility Correlation:

  • Integration of ChIP-seq data with ATAC-seq or DNase-seq

  • Analysis of 2-hydroxyisobutyryl modification enrichment at open chromatin regions

  • Characterization of chromatin state transitions associated with this modification

• Protein Interaction Networks:

  • Identification of reader proteins that specifically recognize 2-hydroxyisobutyryl modifications

  • Characterization of writer and eraser enzymes regulating this modification

  • Mapping of interaction networks associated with modified histones

• Single-Cell Applications:

  • Adaptation of antibodies for single-cell ChIP or CUT&Tag protocols

  • Integration with single-cell transcriptomics and proteomics

  • Characterization of cellular heterogeneity in modification patterns

These integrated approaches provide comprehensive insights into the functional role of 2-hydroxyisobutyryl modifications in chromatin biology and gene regulation, positioning this modification within the broader context of epigenetic regulation mechanisms .