2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody

What is 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody and what epitope does it specifically recognize?

2-Hydroxyisobutyryl-HIST1H2AG (K95) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the 2-hydroxyisobutyryl post-translational modification at lysine 95 (K95) of human Histone H2A type 1 (HIST1H2AG) . This antibody targets a specific peptide sequence surrounding the 2-hydroxyisobutyrylated lysine 95 residue derived from Human Histone H2A type 1 . The antibody belongs to the IgG isotype and is primarily designed for detecting this specific histone modification in human samples .

The antibody's specificity for the 2-hydroxyisobutyryl modification represents an important tool for studying this relatively newer identified post-translational modification in histone biology and epigenetic regulation mechanisms.

What are the validated research applications for 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody?

Based on current validation data, 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody has been successfully tested and validated for the following research applications:

The antibody has demonstrated successful detection of 2-hydroxyisobutyrylated HIST1H2AG in immunofluorescent analysis of HeLa cells, confirming its utility for cellular localization studies . This makes it particularly valuable for researchers investigating nuclear distribution patterns of this specific histone modification.

What are the optimal storage and handling conditions for maintaining antibody activity?

For optimal performance of the 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody, the following storage and handling recommendations should be followed:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Buffer composition: The antibody is supplied in a buffer containing 0.03% Proclin 300 as a preservative and 50% Glycerol

• Appearance: Liquid form

• Shelf-life: 12 months from date of receipt when properly stored

Researchers should avoid repeated freeze-thaw cycles as these can significantly diminish antibody activity. Creating small working aliquots upon first thaw is strongly recommended for preserving antibody function over extended periods.

How does 2-hydroxyisobutyrylation differ from other histone post-translational modifications?

2-Hydroxyisobutyrylation represents a distinct histone post-translational modification that differs from better-characterized modifications like acetylation and methylation:

• While acetylation involves the addition of an acetyl group to lysine residues that neutralizes the positive charge and generally promotes transcriptional activation , 2-hydroxyisobutyrylation involves the addition of a bulkier 2-hydroxyisobutyryl group.

• Unlike methylation, which can occur at different levels (mono-, di-, or tri-methylation) and has variable effects on transcription depending on the specific residue modified , 2-hydroxyisobutyrylation appears to be primarily associated with transcriptionally active regions .

• 2-Hydroxyisobutyrylation of histones appears to be enriched in transcriptionally active chromatin regions, similar to H2A.B variant incorporation .

• Based on emerging research, 2-hydroxyisobutyrylation may work in concert with histone variant dynamics to regulate chromatin accessibility and gene expression .

What is the relationship between HIST1H2AG and other histone H2A variants?

HIST1H2AG is part of the histone H2A family, which includes several variants with distinct functions in chromatin organization:

• HIST1H2AG (also known as H2A.1 or H2A/ptl) is a canonical H2A histone type that, along with H2B, forms the H2A/H2B dimers in nucleosomes .

• The H2A family includes multiple variants with specialized functions, including H2A.B, which is enriched in transcriptionally active and highly dynamic chromatin .

• Unlike the variant H2A.B, which is associated with highly dynamic nucleosomes that yield heterogeneously sized DNA fragments upon nuclease digestion, canonical H2A histones like HIST1H2AG typically form more stable nucleosome structures .

• The UniProt ID for HIST1H2AG is P0C0S8, and it has several alternative names including H2AC11, H2AFP, H2AC13, H2AFC, H2AC15, H2AFD, and others .

What experimental considerations are crucial when validating 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody specificity?

When validating the specificity of 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody, researchers should implement the following critical controls and considerations:

• Peptide Competition Assay: Pre-incubate the antibody with excess synthetic 2-hydroxyisobutyrylated K95 peptide before application in the experimental system. The loss of signal confirms specificity for the modified epitope.

• Cross-reactivity Assessment: Test against similar histone modifications, particularly acetylation at K95, to ensure the antibody distinguishes between different post-translational modifications at the same residue .

• Biological Controls:

  • Positive controls: Use cell types known to have high levels of histone 2-hydroxyisobutyrylation, such as transcriptionally active cells

  • Negative controls: Include samples where the modification is enzymatically removed or cells treated with inhibitors of the enzymatic pathway responsible for 2-hydroxyisobutyrylation

• Technical Controls for Immunofluorescence:

  • Secondary antibody-only controls to assess background

  • Nuclear counterstaining to confirm nuclear localization

  • Recommended dilution range testing (1:50-200) to determine optimal signal-to-noise ratio

• Western Blot Validation: Confirm the antibody detects a band of the expected molecular weight (~14-15 kDa for histone H2A) and shows intensity changes under conditions that alter 2-hydroxyisobutyrylation levels .

These validation steps are essential for ensuring experimental reproducibility and accurate interpretation of results when investigating this specific histone modification.

How can 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody be integrated into chromatin immunoprecipitation (ChIP) protocols?

While the search results don't specifically mention ChIP applications for this antibody, researchers interested in adapting the 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody for ChIP studies should consider the following methodological approach:

• Protocol Optimization:

  • Starting concentration: Test a range of antibody concentrations (2-10 μg per ChIP reaction)

  • Crosslinking time: Optimize formaldehyde crosslinking (typically 10-15 minutes) to preserve the modification

  • Sonication parameters: Adjust to generate 200-500 bp DNA fragments for optimal epitope exposure

• Controls for ChIP-Seq Experiments:

  • Input controls: Use sonicated chromatin before immunoprecipitation

  • IgG controls: Include rabbit IgG as a negative control

  • Positive controls: Include antibodies against known active chromatin marks (H3K4me3, H3K27ac)

• Data Analysis Considerations:

  • Compare 2-hydroxyisobutyryl-K95 enrichment patterns with transcriptionally active regions

  • Examine co-localization with histone variant H2A.B, which is known to be enriched in transcriptionally active chromatin

  • Correlate with RNA-seq data to establish functional relationships with gene expression

• Sequential ChIP (Re-ChIP):

  • Consider sequential immunoprecipitation with antibodies against other histone modifications to identify co-occurring modifications

  • This approach can reveal functional interactions between 2-hydroxyisobutyrylation and other epigenetic marks

What is the role of 2-hydroxyisobutyrylation at K95 in HIST1H2AG in relation to chromatin dynamics and transcriptional regulation?

Recent research suggests that 2-hydroxyisobutyrylation of histones plays a significant role in chromatin dynamics and transcriptional regulation:

• Association with Transcriptionally Active Chromatin:

  • Similar to H2A.B enrichment, 2-hydroxyisobutyrylation appears to be associated with transcriptionally active regions of chromatin

  • The modification may contribute to creating a more accessible chromatin environment, facilitating transcription factor binding and RNA polymerase progression

• Dynamic Chromatin States:

  • Evidence suggests that 2-hydroxyisobutyrylation, particularly in histone H2A variants, may contribute to increased nucleosome dynamics

  • The modification may destabilize nucleosome structure, similar to how H2A.B incorporation results in more dynamic nucleosomes that yield heterogeneous DNA fragment patterns upon nuclease digestion

• Relationship to Other Epigenetic Mechanisms:

  • 2-Hydroxyisobutyrylation likely works in concert with other histone modifications and chromatin remodeling factors

  • The BAP1 deubiquitinase, which regulates H2AK119ub levels, has been shown to influence B-cell activation and humoral immunity , suggesting complex interplay between different histone modifications

• Regulation of Specific Cellular Processes:

  • While direct evidence for K95 2-hydroxyisobutyrylation is limited in the search results, research on histone modifications broadly suggests potential roles in:

    • Immune cell activation and differentiation

    • Response to viral infection, as demonstrated by studies on HSV-1 chromatin dynamics

    • Gene expression programs during cellular development and differentiation

Understanding the specific role of 2-hydroxyisobutyrylation at K95 in HIST1H2AG requires further research, but current evidence suggests it contributes to creating and maintaining transcriptionally permissive chromatin environments.

How can multiplexed imaging approaches be optimized when using 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody?

For researchers implementing multiplexed imaging approaches with 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody, the following methodological considerations are recommended:

• Antibody Compatibility Assessment:

  • Ensure primary antibodies are raised in different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential detection with complete elution between rounds

  • Test for potential epitope masking when multiple histone modification antibodies are used simultaneously

• Fluorophore Selection and Imaging Parameters:

  • Choose fluorophores with minimal spectral overlap

  • For the 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody, follow the validated immunofluorescence protocol with dilutions of 1:50-200

  • Implement appropriate controls for each fluorescence channel

• Sample Preparation Optimization:

  • Cell fixation: Test both paraformaldehyde (PFA) and methanol fixation to determine which best preserves the epitope

  • Permeabilization: Optimize detergent concentration and incubation time for nuclear penetration without destroying epitopes

  • Antigen retrieval: Evaluate if heat-induced or enzymatic antigen retrieval improves signal detection

• Quantitative Analysis Approach:

  • Implement nucleus segmentation algorithms for single-cell analysis

  • Quantify nuclear distribution patterns and co-localization with other chromatin markers

  • Correlate 2-hydroxyisobutyryl-K95 signal intensity with functional readouts (e.g., transcriptional activity markers)

• Documentation and Standardization:

  • Record all imaging parameters (exposure times, gain settings, objectives used)

  • Include all controls in supplementary materials when publishing

  • Consider depositing standardized protocols in repositories for reproducibility

What methodological approaches can be used to study the functional interplay between 2-hydroxyisobutyrylation and variant histone incorporation?

To investigate the functional relationship between 2-hydroxyisobutyryl-HIST1H2AG (K95) and histone variant dynamics (such as H2A.B incorporation), researchers can employ the following methodological approaches:

• Sequential or Dual Immunoprecipitation Studies:

  • Perform ChIP using 2-hydroxyisobutyryl-HIST1H2AG (K95) Antibody followed by immunoprecipitation with antibodies against histone variants

  • This approach can reveal genomic regions where both modifications co-occur

• Genome Editing and Functional Studies:

  • Use CRISPR/Cas9 to mutate K95 to a non-modifiable residue (K95R)

  • Monitor changes in histone variant incorporation and chromatin accessibility

  • Assess effects on transcriptional output using RNA-seq or PRO-seq

• Mass Spectrometry-Based Approaches:

  • Perform quantitative proteomics to measure 2-hydroxyisobutyrylation levels in different histone variant populations

  • Use targeted MS approaches to specifically quantify K95 2-hydroxyisobutyrylation in canonical H2A versus H2A.B

• Cellular Context Manipulation:

  • Study 2-hydroxyisobutyrylation patterns in contexts where H2A.B is known to be enriched, such as:

    • Transcriptionally active regions

    • Dynamic chromatin in viral infections, as observed with HSV-1

    • B cell activation during humoral immune response

• Super-Resolution Microscopy:

  • Implement STORM or STED microscopy to visualize the spatial relationship between 2-hydroxyisobutyrylated histones and specific histone variants at the nanoscale

  • Correlate spatial patterns with functional genomic data

By integrating these approaches, researchers can develop a comprehensive understanding of how 2-hydroxyisobutyrylation at K95 in HIST1H2AG functionally interacts with histone variant dynamics to regulate chromatin structure and function in various biological contexts.