2-hydroxyisobutyryl-HIST1H2BC (K34) Antibody

What is 2-hydroxyisobutyrylation of histones and why is it important in epigenetic research?

2-hydroxyisobutyrylation is a post-translational modification of histones that plays a crucial role in chromatin remodeling and gene transcription regulation. This modification on lysine residues, such as K34 on Histone H2B type 1-C/E/F/G/I (HIST1H2BC), represents an important epigenetic mark that influences chromatin structure and accessibility.

The 2-hydroxyisobutyrylation of HIST1H2BC at lysine 34 is particularly significant as it contributes to the regulatory landscape of gene expression. This histone modification is now recognized as part of the broader "histone code" that determines how genetic information is accessed and expressed. Research using antibodies specific to this modification allows scientists to explore the molecular mechanisms behind gene regulation, cell differentiation, and disease development, making it a valuable target for epigenetic studies .

What applications is the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody validated for?

The 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody has been validated for multiple research applications, with specific recommended dilutions for optimal results:

The antibody has been specifically tested with human samples and demonstrates high specificity for the 2-hydroxyisobutyryl modification at K34 of HIST1H2BC . When designing experiments, researchers should titrate the antibody to determine optimal concentrations for their specific experimental conditions and cell types.

What is the specificity of the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody?

The 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody specifically recognizes the 2-hydroxyisobutyryl modification at lysine 34 on Histone H2B type 1-C/E/F/G/I. The antibody has been generated using a peptide sequence surrounding the 2-hydroxyisobutyryl-Lys (34) site derived from Human Histone H2B type 1-C/E/F/G/I .

The specificity of this antibody is critical for distinguishing between different histone modifications and ensuring accurate experimental results. The polyclonal version of this antibody (such as PAC060484) is raised in rabbits and shows high specificity for human samples . It's important to note that the antibody targets a specific modification site (K34) on a specific histone variant (HIST1H2BC, also known as H2BC4), which is part of the H2B histone family .

For validation experiments, researchers often use cells treated with sodium butyrate (a histone deacetylase inhibitor that can increase certain histone modifications) to confirm antibody specificity, as mentioned in product validation data .

What are the recommended storage conditions for the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody?

Proper storage is essential for maintaining antibody activity and specificity. Based on manufacturer recommendations, the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody should be stored according to these guidelines:

The antibody is typically supplied in liquid form, and some versions may contain additional components like BSA (0.1%) for stability . Long-term storage at recommended temperatures ensures the antibody maintains its specificity and reactivity for extended periods. Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody quality and performance.

How can I optimize Western blot protocols for detecting 2-hydroxyisobutyrylated HIST1H2BC?

Optimizing Western blot protocols for detecting 2-hydroxyisobutyrylated HIST1H2BC requires several specific considerations:

Sample Preparation:

• Extract histones using specialized acid extraction methods to enrich for histone proteins

• Include histone deacetylase inhibitors (e.g., sodium butyrate at 30mM for 4 hours) in cell culture prior to extraction to preserve modifications

• Use freshly prepared samples when possible to maintain modification integrity

Electrophoresis and Transfer:

• Use specialized gel systems designed for low molecular weight proteins (14-17 kDa for Histone H2B)

• Consider gradient gels (12-15%) for better resolution

• Transfer to PVDF membrane at lower voltage for longer time to ensure complete transfer of small proteins

Blocking and Detection:

• Use 5% BSA in TBST rather than milk for blocking (milk contains histones that may cause background)

• Incubate primary antibody (2-hydroxyisobutyryl-HIST1H2BC K34) at 1:100-1:1000 dilution overnight at 4°C

• Include positive controls such as A549 or K562 cell lysates treated with sodium butyrate

• Include total Histone H2B antibody as a loading control on a separate blot

Validation:

• Run side-by-side comparisons with untreated and sodium butyrate-treated samples to demonstrate specificity

• Consider using a pan-2-hydroxyisobutyryl antibody alongside the site-specific K34 antibody for confirmation

This optimized protocol will help ensure specific detection of 2-hydroxyisobutyrylated HIST1H2BC while minimizing background and non-specific signals.

What controls should be incorporated in ChIP experiments using the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody?

For rigorous ChIP experiments using the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody, the following controls are essential:

Positive Controls:

• Input DNA (pre-immunoprecipitation sample) - serves as a reference for enrichment calculations

• ChIP with a general H2B antibody to confirm histone presence

• ChIP with a known actively transcribed gene region where 2-hydroxyisobutyrylation is expected

• Cells treated with HDAC inhibitors (e.g., sodium butyrate) to increase histone modification levels

Negative Controls:

• IgG control from the same species as the primary antibody (rabbit IgG for polyclonal antibodies)

• ChIP with a gene desert region or heterochromatic region with expected low modification levels

• ChIP with peptide competition (using the immunizing peptide) to demonstrate specificity

• Untreated cells as baseline for comparison with treatments that alter 2-hydroxyisobutyrylation

Technical Validation:

• Sequential ChIP (re-ChIP) with a second antibody against active chromatin marks to confirm co-localization

• Western blot verification of the antibody specificity before ChIP

• qPCR of known regulated regions before committing to genome-wide sequencing

• Biological replicates (minimum of 3) to ensure reproducibility

These controls will help validate ChIP results, demonstrate antibody specificity, and provide confidence in genomic distribution patterns of 2-hydroxyisobutyrylation at HIST1H2BC K34.

How does 2-hydroxyisobutyrylation differ from other histone modifications in terms of function and distribution?

2-hydroxyisobutyrylation represents a distinct histone modification with unique characteristics compared to other better-studied modifications:

Functionally, 2-hydroxyisobutyrylation at HIST1H2BC K34 is emerging as an important mark for gene activation, potentially with distinct regulatory roles from other modifications. While acetylation and 2-hydroxyisobutyrylation both neutralize the positive charge of lysine residues, the bulkier 2-hydroxyisobutyryl group may cause more significant structural changes to chromatin and recruit different reader proteins .

Unlike some better-characterized modifications, the complete set of writer, reader, and eraser enzymes for 2-hydroxyisobutyrylation is still being investigated, making antibodies against specific sites like HIST1H2BC K34 crucial tools for advancing this research area.

What is the effect of sodium butyrate treatment on 2-hydroxyisobutyrylation levels?

Sodium butyrate treatment significantly impacts 2-hydroxyisobutyrylation levels through multiple mechanisms:

Experimental Evidence:
Western blot analysis of A549 and K562 whole cell lysates shows a marked increase in 2-hydroxyisobutyryl-HIST1H2BC (K34) signal when treated with 30mM sodium butyrate for 4 hours compared to untreated controls . This treatment paradigm is commonly used as a positive control when validating antibodies against this modification.

Mechanistic Basis:

• Sodium butyrate functions as a histone deacetylase (HDAC) inhibitor, preventing the removal of various acyl modifications from histones

• While primarily known for increasing acetylation, sodium butyrate also impacts other acylation marks including 2-hydroxyisobutyrylation

• The treatment may also influence metabolic pathways that generate 2-hydroxyisobutyryl-CoA, the metabolic substrate for this modification

• Changes in chromatin structure due to increased acetylation may make sites more accessible for 2-hydroxyisobutyrylation

Experimental Applications:
Researchers can use sodium butyrate treatment (20-30mM for 4-6 hours) as:

• A positive control for antibody validation

• A tool to study the dynamics of 2-hydroxyisobutyrylation

• A method to enhance signal in experiments where 2-hydroxyisobutyrylation levels are otherwise low

• A comparative reference to study the relationship between different histone modifications

This treatment approach provides a valuable tool for researchers exploring the biological significance of HIST1H2BC K34 2-hydroxyisobutyrylation in various cellular contexts.

What are the optimal fixation and permeabilization methods for immunofluorescence using the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody?

Optimizing fixation and permeabilization is critical for successful immunofluorescence (IF) detection of 2-hydroxyisobutyryl-HIST1H2BC (K34):

Recommended Fixation Protocols:

Permeabilization Optimization:

• For PFA-fixed cells: 0.1-0.2% Triton X-100 in PBS for 10 minutes at room temperature

• Careful titration of permeabilization time is critical - excessive permeabilization can lead to signal loss

• Alternative: 0.5% saponin may provide gentler permeabilization with less epitope disruption

Antigen Retrieval Considerations:

• Heat-mediated antigen retrieval in citrate buffer (pH 6.0) may enhance signal for some samples

• For tissue sections, test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for optimal results

Protocol Optimization:

• For the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody, use at 1:1-1:10 dilution in IF applications

• Include 1% BSA in antibody dilution buffer to reduce background

• Extended primary antibody incubation (overnight at 4°C) often yields better results

• HeLa cells have been validated as a positive control for IF applications

These optimized protocols will help ensure specific detection of the 2-hydroxyisobutyryl modification while maintaining cellular architecture for accurate localization studies.

How can I troubleshoot non-specific binding with the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody?

Non-specific binding is a common challenge when working with histone modification antibodies. Here's a comprehensive troubleshooting approach for the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody:

Common Issues and Solutions:

Validation Approaches:

• Peptide Competition Assay: Pre-incubate antibody with increasing concentrations of the immunizing peptide before application to demonstrate specificity

• Knockout/Knockdown Controls: If available, use samples with reduced HIST1H2BC expression as negative controls

• Dilution Series: Test a range of antibody dilutions to determine optimal signal-to-noise ratio

• Alternative Detection Methods: Confirm findings using a different method (e.g., validate WB findings with IF)

• Treatment Controls: Compare untreated samples with those treated with sodium butyrate to confirm proper detection of 2-hydroxyisobutyrylation

Advanced Troubleshooting:

• Antigen retrieval optimization: Test different pH conditions and retrieval methods

• Secondary antibody controls: Run experiments with secondary antibody only to identify non-specific binding

• Sample preparation refinement: Consider specialized histone extraction protocols to enhance purity

Implementing these troubleshooting strategies will help ensure specific detection of 2-hydroxyisobutyryl-HIST1H2BC (K34) and improve experimental reproducibility.

How can I quantify changes in 2-hydroxyisobutyrylation in response to metabolic or cellular stress?

Accurate quantification of 2-hydroxyisobutyrylation changes requires robust methodological approaches:

Western Blot Quantification:

• Treat cells with relevant stressors (oxidative stress, nutrient deprivation, hypoxia)

• Extract histones using acid extraction methods to enrich for histone proteins

• Run equal amounts of histone extracts on SDS-PAGE

• Probe with 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody (1:100-1:1000 dilution)

• Normalize to total H2B levels using a pan-H2B antibody on a separate blot

• Use digital imaging and analysis software (ImageJ, Li-COR) for densitometry

• Calculate fold change relative to control conditions

ELISA-Based Approaches:

• Develop a sandwich ELISA using the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody (1:2000-1:10000)

• Generate a standard curve using known quantities of modified peptide

• Analyze histone extracts from control and stressed cells

• Normalize to total protein or total H2B levels

Mass Spectrometry for Comprehensive Analysis:

• Perform histone extraction and propionylation of unmodified lysines

• Digest with trypsin and enrich for modified peptides

• Analyze by LC-MS/MS with multiple reaction monitoring

• Quantify the ratio of modified to unmodified peptides containing K34

• Compare relative abundances across treatment conditions

Flow Cytometry for Single-Cell Analysis:

• Fix and permeabilize cells using optimized protocols

• Stain with 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody (0.40 μg per 10^6 cells)

• Analyze fluorescence intensity as a measure of modification levels

• Gate on specific cell populations to assess cell cycle-dependent changes

• Compare median fluorescence intensity across conditions

These methodological approaches provide complementary data on how 2-hydroxyisobutyrylation levels respond to various cellular stressors, allowing researchers to investigate the biological significance of this modification in stress responses.

What genomic regions are enriched for 2-hydroxyisobutyrylation and how does this compare to other histone modifications?

The genomic distribution of 2-hydroxyisobutyrylation presents a distinctive pattern with important implications for gene regulation:

Genomic Distribution Patterns:

Experimental Approaches to Map Distribution:

• ChIP-seq Analysis: Using the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody for chromatin immunoprecipitation followed by next-generation sequencing

• CUT&RUN: For higher resolution mapping with lower background

• Comparative Analysis: Side-by-side comparison with ChIP-seq data for other modifications (acetylation, methylation)

• Integration with RNA-seq: Correlation of modification location with gene expression data

Biological Implications:
The distinct distribution pattern of 2-hydroxyisobutyrylation suggests it may play specialized roles in gene regulation beyond those of better-studied modifications. The enrichment at specific genomic regions indicates potential roles in:

• Rapid gene activation responses

• Cell-type specific gene expression patterns

• Metabolic regulation of transcription

• Stress-responsive gene regulation

Understanding the genomic distribution of 2-hydroxyisobutyrylation on HIST1H2BC K34 provides critical insights into its functional significance in chromatin biology and gene expression regulation. Ongoing research using the 2-hydroxyisobutyryl-HIST1H2BC (K34) antibody continues to refine our understanding of this emerging epigenetic mark.