2-hydroxyisobutyryl-HIST1H2AG (K5) Antibody

What is 2-hydroxyisobutyryl-HIST1H2AG (K5) Antibody and how does it differ from other residue-specific variants?

The 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody is a rabbit polyclonal primary antibody that specifically recognizes 2-hydroxyisobutyrylation at the lysine 5 residue of histone H2A type 1 protein . This antibody is generated using a synthesized peptide derived from Human Histone H2A type 1 protein (2-14aa) as the immunogen . It is important to distinguish this antibody from similar variants that target different lysine residues on the same protein, such as the K9 variant (which recognizes 2-hydroxyisobutyrylation at lysine 9) and the K74 variant (which targets lysine 74) . Each of these antibodies provides specific information about different modification sites that may have distinct biological functions and regulatory roles in chromatin dynamics. The K5 antibody specifically allows researchers to study modifications at a location that may be involved in unique chromatin regulatory events.

What are the technical specifications researchers should know about this antibody?

The 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody is available as an unconjugated rabbit polyclonal antibody with IgG isotype . Key specifications include:

This antibody recognizes the human HIST1H2AG protein, which has multiple synonyms including H2AC11, H2A.1, and Histone H2A type 1 . Understanding these specifications is essential for proper experimental design and interpretation of results when working with this antibody.

Why study 2-hydroxyisobutyrylation of histones in research applications?

2-hydroxyisobutyrylation represents a relatively newly discovered histone post-translational modification that plays important roles in chromatin structure and gene regulation. Histones, particularly H2A (which includes HIST1H2AG), are nuclear proteins crucial for maintaining chromosome structure in eukaryotes . The nucleosome consists of approximately 146bp of DNA wrapped around a histone octamer composed of H2A, H2B, H3, and H4 . H2A specifically ensures proper assembly and functioning of the centromere and contributes significantly to gene regulation . The specific modification at lysine 5 (K5) may have distinct regulatory functions compared to modifications at other lysine residues (like K9 or K74). By using residue-specific antibodies like the 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody, researchers can investigate site-specific effects of this modification on chromatin dynamics and gene expression regulation.

What are the recommended applications and optimal protocols for using this antibody?

The 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody has been validated for several applications with specific recommended protocols:

For ELISA:
The antibody has been tested in indirect ELISA applications . While specific dilution ratios for ELISA are not directly provided in the search results for the K5 antibody, similar antibodies targeting different residues suggest working dilutions in the range of 1:100-1:1000 .

For Immunocytochemistry (ICC):
The K5 antibody is suitable for ICC applications . Based on similar antibodies targeting different residues, recommended dilution ranges of 1:20-1:200 are typical .

For Western Blot (WB):
Although not specifically confirmed for the K5 antibody in the search results, related antibodies suggest dilution ranges of 1:100-1:5000 for WB applications . Optimization is recommended for specific experimental conditions.

For Immunofluorescence (IF):
Similar antibodies are used at dilutions of 1:50-1:200 , which may serve as a starting point for the K5 antibody if used for IF, though specific validation would be necessary.

These recommendations serve as starting points, and researchers should optimize conditions for their specific experimental systems and sample types.

How should researchers optimize storage conditions to maintain antibody efficacy?

To maintain optimal antibody efficacy, the 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody should be stored according to these guidelines:

• Upon receipt, store the antibody at -20°C or -80°C (preferred for long-term storage) .

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce specificity .

• The antibody is supplied in liquid form with a preservative buffer (PBS with 50% glycerol and 0.03% ProClin 300; pH 7.4) , which helps maintain stability.

• For working solutions, aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles.

• When thawing, allow the antibody to come to room temperature slowly and mix gently by inversion rather than vortexing.

Proper storage is crucial for maintaining antibody performance across multiple experiments and ensuring reproducibility of results in epigenetic studies targeting histone modifications.

What control samples and validation methods should be included when using this antibody?

To ensure reliable and interpretable results, researchers should implement the following controls and validation methods:

• Positive controls: Include samples known to express HIST1H2AG with 2-hydroxyisobutyrylation at K5, such as specific human cell lines where this modification has been documented.

• Negative controls:

  • Samples treated with deacetylase/de-2-hydroxyisobutyrylase enzymes

  • Non-human samples (as the antibody specifically reacts with human samples)

  • Primary antibody omission controls

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm specificity.

• Cross-reactivity testing: Validate that the antibody specifically recognizes K5 modification and not K9 or K74 modifications on the same protein.

• Knockout/knockdown validation: If possible, use genetic models where HIST1H2AG expression is reduced to confirm antibody specificity.

• Multiple technique validation: Confirm findings using complementary techniques (e.g., mass spectrometry) to verify the presence and location of the 2-hydroxyisobutyryl modification.

Proper validation ensures that experimental observations genuinely reflect the biology of 2-hydroxyisobutyrylation rather than artifacts or non-specific binding.

How can researchers distinguish between different lysine modifications on the same histone protein?

Distinguishing between different lysine modifications on histone proteins requires careful experimental design and multiple validation approaches:

• Site-specific antibodies: Use antibodies that specifically target different modification sites (e.g., K5 vs. K9 vs. K74) on HIST1H2AG . The antibodies in the search results provide this capability, as they are designed to recognize 2-hydroxyisobutyrylation at specific lysine residues.

• Mass spectrometry analysis: Employ high-resolution mass spectrometry to identify and quantify site-specific modifications. This technique can provide unambiguous identification of modification sites when antibody cross-reactivity is a concern.

• Sequential immunoprecipitation: Perform sequential ChIP experiments using antibodies against different modifications to determine which modifications co-occur on the same nucleosomes.

• Peptide arrays: Use synthetic peptide arrays containing various modified lysine residues to test antibody specificity and potential cross-reactivity between sites.

• Mutational analysis: Utilize site-specific mutations of lysine residues (K→R substitutions) to validate antibody specificity in cellular or reconstituted systems.

These approaches allow researchers to build a comprehensive map of histone modifications and understand how different lysine residues on the same histone protein may have distinct regulatory functions.

What are the most effective experimental designs for studying the temporal dynamics of histone 2-hydroxyisobutyrylation?

To effectively study temporal dynamics of histone 2-hydroxyisobutyrylation, researchers should consider these experimental approaches:

• Time-course experiments: Collect samples at multiple time points after a stimulus (e.g., cell cycle progression, differentiation signals, metabolic changes) and analyze changes in 2-hydroxyisobutyrylation levels using the appropriate antibody.

• Pulse-chase experiments: Use metabolic labeling of 2-hydroxyisobutyryl groups followed by chase periods to determine the turnover rates of this modification.

• Live-cell imaging: Although challenging, developing fluorescent sensors for 2-hydroxyisobutyrylation could enable real-time monitoring of modification dynamics.

• Synchronized cell populations: Use cell synchronization methods to study modification patterns at specific cell cycle phases.

• ChIP-seq time series: Perform ChIP-seq with the 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody at different time points to map genome-wide changes in modification patterns.

• Integrated multi-omics approaches: Combine histone modification analysis with transcriptomics and metabolomics to correlate 2-hydroxyisobutyrylation changes with gene expression and metabolic states.

These approaches help researchers understand not just where 2-hydroxyisobutyrylation occurs, but how its dynamic regulation contributes to cellular processes and responses to environmental changes.

How does 2-hydroxyisobutyrylation interact with other histone modifications in the histone code?

Understanding the interplay between 2-hydroxyisobutyrylation and other histone modifications requires sophisticated experimental approaches:

• Sequential ChIP (Re-ChIP): Perform immunoprecipitation with the 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody followed by a second immunoprecipitation with antibodies against other modifications to identify co-occurrence patterns.

• Mass spectrometry-based proteomics: Use high-resolution mass spectrometry to identify combinatorial modification patterns on histone peptides, revealing which modifications co-exist or are mutually exclusive.

• Protein interaction studies: Identify reader proteins that specifically recognize 2-hydroxyisobutyrylated histones and determine if their binding is affected by neighboring modifications.

• Enzymatic studies: Investigate how the presence of one modification affects the addition or removal of others by using in vitro enzymatic assays with modified histone substrates.

• Genomic colocalization analysis: Perform ChIP-seq with antibodies against multiple modifications to map their genome-wide distribution patterns and identify regions of overlap or mutual exclusion.

• Functional genomics screens: Use CRISPR screens targeting various histone-modifying enzymes to uncover functional relationships between different modifications.

What are common technical challenges when using 2-hydroxyisobutyryl-HIST1H2AG antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with histone modification-specific antibodies. Here are common issues and solutions:

• Non-specific binding:

  • Problem: Background signal or multiple bands in Western blots

  • Solution: Optimize blocking conditions (try 5% BSA instead of milk), increase washing stringency, and titrate antibody concentration. Use the recommended dilution ranges (e.g., 1:100-1:1000 for WB) .

• Cross-reactivity between modification sites:

  • Problem: Antibody recognizing 2-hydroxyisobutyrylation at sites other than K5

  • Solution: Perform peptide competition assays with modified and unmodified peptides to confirm specificity. Validate results using mass spectrometry or other site-specific methods.

• Signal variability:

  • Problem: Inconsistent results between experiments

  • Solution: Standardize sample preparation, avoid repeated freeze-thaw cycles of the antibody , and include appropriate positive controls in each experiment.

• Epitope masking:

  • Problem: Reduced signal due to fixation or other treatments

  • Solution: Optimize fixation conditions and consider antigen retrieval methods for formaldehyde-fixed samples.

• Low signal-to-noise ratio in ICC:

  • Problem: Weak specific signal relative to background

  • Solution: Start with more concentrated antibody dilutions (e.g., 1:20-1:200 for ICC) and optimize incubation times and temperatures.

Careful optimization of experimental conditions and inclusion of appropriate controls can address most technical challenges encountered with these antibodies.

How can researchers ensure reproducibility when using 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody across different batches?

Ensuring reproducibility when working with antibodies targeting histone modifications requires systematic approaches:

• Batch validation:

  • Document lot numbers and test each new batch against a reference sample

  • Maintain a consistent positive control sample across experiments

• Standardized protocols:

  • Develop detailed SOPs for each application (ELISA, WB, ICC)

  • Record all experimental conditions, including incubation times, temperatures, and buffer compositions

• Internal controls:

  • Include loading controls for total histone H2A

  • Use spike-in standards when possible

• Quantification methods:

  • Adopt consistent quantification methods and software

  • Use relative quantification rather than absolute values when comparing across batches

• Storage consistency:

  • Store all antibody aliquots under identical conditions (-20°C or -80°C)

  • Maintain consistent thawing procedures

• Documentation:

  • Maintain detailed records of antibody performance across experiments

  • Document any batch-specific optimizations required

By implementing these practices, researchers can minimize batch-to-batch variability and ensure consistent, reliable results when studying histone 2-hydroxyisobutyrylation patterns.

What are the key considerations for quantitative analysis of 2-hydroxyisobutyrylation levels?

Accurate quantification of 2-hydroxyisobutyrylation levels requires careful consideration of several factors:

• Normalization strategies:

  • Normalize to total histone levels using pan-histone antibodies

  • Consider multiple housekeeping proteins rather than relying on a single control

  • For ChIP-seq analysis, use spike-in controls for between-sample normalization

• Dynamic range limitations:

  • Establish a standard curve to ensure measurements fall within the linear range of detection

  • Perform serial dilutions of samples to confirm linearity of the assay

• Statistical approaches:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological replicates (n≥3) rather than technical replicates alone

  • Account for batch effects in experimental design and analysis

• Technical considerations:

  • For Western blots, use fluorescent secondary antibodies for more accurate quantification

  • For ELISA, ensure consistent plate preparation and development times

  • For ICC/IF, standardize image acquisition parameters and analysis protocols

• Comparative analysis:

  • When comparing different lysine modifications (K5 vs. K9 vs. K74), account for potential differences in antibody affinity

  • Consider orthogonal methods (e.g., mass spectrometry) for absolute quantification

Addressing these considerations ensures that quantitative analyses of 2-hydroxyisobutyrylation accurately reflect biological reality rather than technical artifacts.

How might researchers integrate 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody into multi-omics approaches?

Integration of histone modification analyses into multi-omics approaches represents a frontier in epigenetic research:

• ChIP-seq integration:

  • Perform ChIP-seq using the 2-hydroxyisobutyryl-HIST1H2AG (K5) antibody to map genome-wide distribution

  • Integrate with RNA-seq data to correlate modification patterns with gene expression

  • Combine with ATAC-seq to understand relationships between this modification and chromatin accessibility

• Proteomics connections:

  • Use antibody-based enrichment followed by mass spectrometry to identify proteins that interact with 2-hydroxyisobutyrylated histones

  • Develop proximity labeling approaches to identify readers of this modification in living cells

• Metabolomics links:

  • Investigate connections between cellular metabolism and 2-hydroxyisobutyrylation levels

  • Trace isotope-labeled metabolic precursors to understand the dynamics of modification turnover

• Single-cell approaches:

  • Adapt antibody-based detection for single-cell epigenomic profiling

  • Correlate single-cell transcriptomics with histone modification patterns

• Spatial epigenomics:

  • Develop in situ approaches using the antibody to map modification patterns within the nucleus

  • Integrate with spatial transcriptomics to understand territorial organization of modified chromatin and gene expression

These integrated approaches will provide a more holistic understanding of how 2-hydroxyisobutyrylation contributes to genome regulation in different cellular contexts and disease states.

What emerging technologies might enhance the study of site-specific histone modifications beyond antibody-based detection?

While antibodies remain valuable tools, emerging technologies offer new possibilities for studying site-specific histone modifications:

• CRISPR-based epigenome editing:

  • Develop targeted approaches to induce or remove 2-hydroxyisobutyrylation at specific genomic loci

  • Engineer reader domains that specifically recognize 2-hydroxyisobutyrylated K5 for targeted modulation

• Nanopore sequencing:

  • Direct detection of histone modifications through nanopore technology

  • Long-read approaches to map combinatorial modification patterns on individual nucleosomes

• Advanced imaging:

  • Super-resolution microscopy combined with specific antibodies to visualize modification patterns at nanoscale resolution

  • Live-cell sensors for dynamic tracking of modification states

• Chemical biology approaches:

  • Click chemistry-based labeling of specific modifications for visualization and enrichment

  • Photocrosslinking strategies to capture transient interactions with modified histones

• Computational predictions:

  • Machine learning models to predict modification sites based on DNA sequence and other epigenetic features

  • Integrative modeling of histone modification networks

These emerging technologies will complement antibody-based approaches and provide new insights into the biology of histone 2-hydroxyisobutyrylation that current methods cannot achieve.

What are the implications of 2-hydroxyisobutyryl-HIST1H2AG (K5) research for understanding disease mechanisms?

Understanding the role of 2-hydroxyisobutyrylation in disease contexts represents an important frontier in epigenetics research:

• Cancer biology:

  • Investigate whether altered patterns of 2-hydroxyisobutyrylation at K5 are associated with specific cancer types

  • Determine if these modifications can serve as biomarkers or therapeutic targets

• Metabolic disorders:

  • Explore connections between metabolic dysregulation and changes in histone 2-hydroxyisobutyrylation patterns

  • Investigate how diet and lifestyle factors influence this modification

• Neurodegenerative diseases:

  • Study the stability of this modification in aging neurons and its potential role in age-related cognitive decline

  • Determine if altered 2-hydroxyisobutyrylation contributes to pathological protein aggregation

• Developmental disorders:

  • Examine the role of 2-hydroxyisobutyrylation in cell fate decisions during development

  • Investigate whether dysregulation of this modification contributes to developmental abnormalities

• Immunological conditions:

  • Study how 2-hydroxyisobutyrylation patterns change during immune cell activation and differentiation

  • Determine if these modifications influence inflammatory responses

By investigating these disease connections, researchers can potentially identify new therapeutic targets and develop epigenetic biomarkers based on site-specific histone modifications like 2-hydroxyisobutyrylation at K5 of HIST1H2AG.