2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody

What is 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody and what does it detect?

2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody (PACO65130) is a polyclonal antibody produced in rabbits that specifically recognizes the 2-hydroxyisobutyryl modification at lysine 108 of Histone H2B type 1-C/E/F/G/I protein. This antibody is designed for studying this specific histone modification, which plays a role in gene regulation and chromatin structure . Unlike general histone antibodies, this targets the specific post-translational modification at a precise amino acid position (K108) within the histone variant HIST1H2BC .

How does 2-hydroxyisobutyryl-HIST1H2BC (K108) modification differ from other histone modifications?

The 2-hydroxyisobutyryl modification is a relatively newly discovered histone modification compared to more well-studied marks like acetylation or methylation. It involves the addition of a 2-hydroxyisobutyryl group to the lysine residue at position 108 on the HIST1H2BC protein. This specific modification contributes to the histone code that regulates chromatin structure and gene expression . Research indicates that 2-hydroxyisobutyrylation likely functions distinctly from acetylation in regulating cellular processes, as evidenced by its presence in different chromatin contexts and its association with specific cellular states .

What research applications is the 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody validated for?

The 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody has been validated for several research applications, primarily:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunocytochemistry (ICC)

For these applications, the recommended dilutions are:

• ICC: 1:10-1:100

When establishing protocols, researchers should perform optimization experiments to determine the optimal dilution for their specific experimental conditions, considering factors such as sample type, fixation method, and detection system.

What experimental controls should be included when using this antibody?

When designing experiments with the 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody, proper controls are essential for result validation:

• Positive control: Use cell lines known to express the 2-hydroxyisobutyryl modification at HIST1H2BC K108, such as certain human cancer cell lines (e.g., A549, K562, or HepG2 treated with sodium butyrate)

• Negative control:

  • Omission of primary antibody

  • Samples where the modification has been enzymatically removed

  • Non-reactive species samples (as the antibody is specific to human samples)

• Specificity control: Pre-incubation of the antibody with the immunizing peptide to demonstrate binding specificity

• Loading control: Use of pan-histone H2B antibodies to normalize for total H2B levels

Proper controls are critical as inadequate antibody characterization is a major source of irreproducibility in biomedical research, with an estimated 50% of commercial antibodies failing to meet basic characterization standards .

How can I verify the specificity of 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody against other histone variants?

Verifying antibody specificity against highly similar histone variants requires a multi-faceted approach:

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of the specific immunizing peptide (Human Histone H2B type 1-C/E/F/G/I protein residues 104-115aa) versus peptides containing 2-hydroxyisobutyryl modifications at other lysine residues (e.g., K120).

• Knockout/knockdown validation: Use CRISPR-Cas9 or siRNA to deplete the target histone variant or the enzymes responsible for the 2-hydroxyisobutyryl modification.

• Mass spectrometry validation: Compare antibody-based detection with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm the presence and location of the modification.

• Cross-reactivity testing: Test the antibody against recombinant histones with various modifications to assess potential cross-reactivity with other variants or modifications (particularly important to distinguish between HIST1H2BC, HIST1H2BE, HIST1H2BF, HIST1H2BG, and HIST1H2BI, which share high sequence homology) .

This comprehensive validation is essential as the highly conserved nature of histone variants makes distinguishing between similar modifications challenging .

What is the relationship between DNA methylation and 2-hydroxyisobutyryl-HIST1H2BC expression in cancer cell models?

Research in endocrine-resistant breast cancer models has revealed important relationships between DNA methylation and histone variants:

• Methylation status: Studies have shown that the highly related histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant C4-12 and long-term estrogen-deprived (LTED) cells compared to parental MCF-7 cells .

• Expression correlation: Hypomethylation of histone H2B variants correlates with increased expression in resistant cell lines. For example, HIST1H2BE hypomethylation overlaps with a CpG island in its exonic region and results in complete lack of methylation in resistant cell lines .

• Variant-specific effects: Interestingly, significant overexpression in resistant breast cancer cell lines was observed specifically for HIST1H2BE and not uniformly across related variants (H2BC/F/G/I) .

• Tissue-specific expression: Expression analysis shows that H2B variants display tissue-specific expression patterns, with some variants being more prominently expressed in specific tissues (e.g., testes and thymus for HIST1H2BE) .

When investigating the role of 2-hydroxyisobutyryl-HIST1H2BC (K108) in cancer models, researchers should consider both the methylation status of the gene and the specific expression patterns of this variant compared to related H2B variants.

How can I optimize ChIP-seq protocols specifically for 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody?

Optimizing ChIP-seq for 2-hydroxyisobutyryl-HIST1H2BC (K108) requires specific considerations:

• Crosslinking optimization:

  • Test multiple formaldehyde concentrations (0.75-2%)

  • Evaluate different crosslinking times (5-20 minutes)

  • Consider dual crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde for improved capture

• Sonication parameters:

  • Target chromatin fragments of 200-400bp

  • Optimize sonication conditions to preserve the epitope while achieving appropriate fragmentation

  • Verify fragment size by agarose gel electrophoresis

• Antibody specificity:

  • Perform preliminary ChIP-qPCR at known targets before proceeding to sequencing

  • Include appropriate controls such as IgG and input samples

  • Consider spike-in controls for normalization

• Washing stringency:

  • Optimize salt concentrations in wash buffers to maintain specificity

  • Test detergent concentrations to reduce background

• Library preparation:

  • When preparing sequencing libraries, account for typically lower yields with histone variant-specific antibodies

  • Consider using specialized library preparation kits designed for low-input samples

Remember that validation of ChIP-seq results is critical, ideally through orthogonal methods or by comparing with published datasets of related histone modifications .

What are the optimal storage and handling conditions for 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody?

Proper storage and handling are crucial for maintaining antibody functionality:

Following these guidelines will help maintain antibody performance and extend its useful life . Prior to use, centrifuge the vial briefly to collect contents at the bottom of the tube and ensure consistency in experiments.

What sample preparation techniques optimize detection of 2-hydroxyisobutyryl-HIST1H2BC (K108) in various applications?

Optimal sample preparation varies by application:

For Western Blotting:

• Cell lysis: Use RIPA buffer supplemented with histone deacetylase inhibitors (e.g., sodium butyrate, 30mM) and protease inhibitors to preserve modifications

• Histone extraction: Acid extraction methods (e.g., 0.2N HCl) can improve histone yield and purity

• Sample treatment: Treatment with sodium butyrate for 4 hours can enhance detection of histone modifications, as demonstrated in A549, K562, and HepG2 cell lines

• Loading: 20-40μg of total protein per lane is typically sufficient

For Immunocytochemistry:

• Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilization: 0.1-0.5% Triton X-100 for 5-10 minutes

• Antigen retrieval: Test heat-mediated (citrate buffer, pH 6.0) and enzymatic methods to determine optimal approach

• Blocking: 5% BSA or 10% serum from the species of the secondary antibody

For cell culture experiments, synchronizing cells or manipulating their metabolic state can enhance detection of specific histone modifications based on their cell-cycle or metabolic dependencies.

How can I quantitatively analyze 2-hydroxyisobutyryl-HIST1H2BC (K108) levels in different cell types?

Quantitative analysis of 2-hydroxyisobutyryl-HIST1H2BC (K108) requires rigorous methodology:

• Western blot quantification:

  • Use digital imaging systems with linear dynamic range

  • Normalize to total H2B levels using a pan-H2B antibody

  • Include standard curves of recombinant proteins when possible

  • Analyze band intensity using ImageJ or similar software

• ELISA-based quantification:

  • Develop a sandwich ELISA using anti-H2B capture antibody and anti-2-hydroxyisobutyryl detection antibody

  • Create standard curves using synthetic modified peptides

  • Ensure samples fall within the linear range of detection

• Mass spectrometry:

  • For absolute quantification, use isotope-labeled synthetic peptides as internal standards

  • Multiple reaction monitoring (MRM) can provide precise quantification of specific modifications

  • Consider sample fractionation to enrich for histones before analysis

• Flow cytometry:

  • For cell-type specific analysis in heterogeneous populations

  • Combine with cell surface markers to identify specific populations

  • Use median fluorescence intensity (MFI) for quantitative comparison

Why might I observe inconsistent staining patterns with 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody in immunocytochemistry?

Inconsistent immunocytochemistry staining can result from several factors:

• Epitope accessibility issues:

  • The 2-hydroxyisobutyryl modification may be masked by chromatin compaction or protein-protein interactions

  • Solution: Test different fixation methods and include antigen retrieval steps

• Cell cycle variation:

  • Histone modifications can vary throughout the cell cycle

  • Solution: Synchronize cells or co-stain with cell cycle markers

• Technical variability:

  • Inconsistent fixation times or reagent penetration

  • Solution: Standardize protocols and processing times

• Biological variation:

  • The 2-hydroxyisobutyryl modification at K108 may not be uniformly distributed across all nuclei

  • Solution: Quantify staining across multiple cells and experiments

• Antibody batch variation:

  • Different lots may have subtle differences in specificity or sensitivity

  • Solution: Validate each new lot against previously successful experiments

When optimizing, consider that HIST1H2BE (a related variant) shows tissue-specific expression patterns, being highly expressed in testes and thymus but low in liver , suggesting that detection of 2-hydroxyisobutyryl-HIST1H2BC may similarly vary across cell types.

How can I distinguish between different histone H2B variants when using 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody?

Distinguishing between highly similar histone H2B variants requires specialized approaches:

• Combined immunoprecipitation and mass spectrometry:

  • Perform IP with the 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody

  • Analyze precipitated proteins by LC-MS/MS to identify the exact histone variants present

  • Look for unique peptides that distinguish between variants

• RNA expression analysis:

  • Measure mRNA levels of different H2B variants (HIST1H2BC, HIST1H2BE, HIST1H2BF, HIST1H2BG, HIST1H2BI)

  • Use variant-specific primers as described in the literature

  • Correlate mRNA expression with protein detection

• Sequential immunolabeling:

  • Use variant-specific antibodies in sequential immunolabeling experiments

  • Compare localization patterns to identify differential distribution

• Genetic approaches:

  • Selectively deplete specific variants using siRNA or CRISPR-Cas9

  • Observe changes in antibody staining patterns

Research has shown that despite identical amino acid sequences, variants like HIST1H2BE, HIST1H2BC, HIST1H2BF, HIST1H2BG, and HIST1H2BI show differential expression patterns in different tissues and cell states , making it important to consider which variants might be predominantly expressed in your experimental system.

What strategies can overcome weak signal issues when using 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody?

Addressing weak signal problems requires systematic optimization:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) can enhance sensitivity 10-100 fold

  • Use biotin-streptavidin systems for signal enhancement

  • Consider polymer-based detection systems with multiple secondary antibodies

• Sample enrichment:

  • Perform histone extraction to enrich for target proteins

  • Use subcellular fractionation to isolate nuclear components

  • Consider treatments that might increase the modification (e.g., sodium butyrate treatment)

• Antibody optimization:

  • Test different antibody concentrations beyond the recommended range

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize blocking conditions to reduce background while preserving signal

• Modification enhancement:

  • Inhibit histone deacetylases to enhance detection of histone modifications

  • Modulate cellular metabolism to potentially increase 2-hydroxyisobutyrylation

• Imaging optimization:

  • Use confocal microscopy with appropriate filter settings

  • Optimize exposure times and detector sensitivity

  • Consider super-resolution techniques for detailed localization studies

Remember that variant proteins typically represent small portions of the total cellular histone pool, which can make detection challenging even with specific antibodies .

How can 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody be used to study epigenetic changes in cancer progression?

The 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody can provide valuable insights into cancer epigenetics:

• Comparative profiling:

  • Compare 2-hydroxyisobutyryl-HIST1H2BC (K108) levels between normal and cancer tissues

  • Analyze changes across cancer progression stages

  • Correlate with other epigenetic marks to identify patterns

• Drug response studies:

  • Monitor changes in modification levels following treatment with epigenetic drugs

  • Assess whether 2-hydroxyisobutyrylation can serve as a biomarker for treatment response

  • Investigate relationships with treatment resistance

• Mechanistic investigations:

  • Identify proteins that interact specifically with 2-hydroxyisobutyryl-HIST1H2BC

  • Study the enzymes responsible for adding and removing this modification

  • Investigate downstream effects on gene expression

• Clinical correlations:

  • Develop tissue microarray analyses to correlate modification levels with patient outcomes

  • Investigate potential diagnostic or prognostic applications

Research on related histone H2B variants has already demonstrated their altered expression in endocrine-resistant breast cancer models, with specific variants like HIST1H2BE showing hypomethylation and increased expression in resistant cell lines . Similar investigations with 2-hydroxyisobutyryl-HIST1H2BC (K108) could reveal important cancer-specific patterns.

What are the current limitations in our understanding of 2-hydroxyisobutyryl-HIST1H2BC (K108) function?

Several knowledge gaps exist regarding 2-hydroxyisobutyryl-HIST1H2BC (K108):

• Enzymatic regulation:

  • The specific writers (enzymes that add) and erasers (enzymes that remove) of the 2-hydroxyisobutyryl modification at K108 remain incompletely characterized

  • The regulatory pathways controlling these enzymes are still being elucidated

• Functional significance:

  • The precise role of this modification in gene regulation remains unclear

  • How it differs functionally from other histone modifications (like acetylation) at the same position needs further investigation

• Tissue specificity:

  • While research has revealed tissue-specific expression patterns of histone variants , the tissue-specific patterns of 2-hydroxyisobutyrylation are less understood

  • The functional significance of these patterns remains to be determined

• Disease associations:

  • Beyond initial observations in cancer models, comprehensive understanding of its role in other diseases is lacking

  • How this modification changes during development and aging needs further study

• Cross-talk with other modifications:

  • The interplay between 2-hydroxyisobutyrylation and other histone modifications in the histone code is not fully mapped

  • How it affects chromatin structure and accessibility needs further characterization

These limitations represent important research opportunities for investigators using the 2-hydroxyisobutyryl-HIST1H2BC (K108) Antibody in their studies.