2-hydroxyisobutyryl-HIST1H2BC (K20) Antibody

What is the 2-hydroxyisobutyryl-HIST1H2BC (K20) Antibody and what is its target?

The 2-hydroxyisobutyryl-HIST1H2BC (K20) Antibody is a polyclonal antibody raised in rabbits that specifically targets the 2-hydroxyisobutyryl modification at lysine 20 of the Hist1H2BC histone protein. This antibody recognizes a specific post-translational modification in histone proteins that are responsible for packaging DNA into chromatin structure. The specific immunogen used is a peptide sequence around the site of 2-hydroxyisobutyryl-Lys (20) derived from Human Histone H2B type 1-C/E/F/G/I .

The antibody enables researchers to detect and analyze 2-hydroxyisobutyrylation, which is a relatively newly characterized histone modification that plays important roles in gene regulation. By binding specifically to this modification site, the antibody allows visualization and quantification of this epigenetic mark in experimental systems .

What are the validated applications for this antibody?

The 2-hydroxyisobutyryl-HIST1H2BC (K20) Antibody has been validated for several experimental applications commonly used in epigenetic and molecular biology research:

The antibody has been specifically tested and verified with human samples, demonstrating its reliability in detecting the target modification in human cell lysates including HeLa, 293, A549, and K562 cell lines, particularly when treated with sodium butyrate to enhance histone modifications .

How does 2-hydroxyisobutyrylation differ from other histone modifications?

2-hydroxyisobutyrylation is a distinct post-translational modification that differs from more commonly studied modifications such as acetylation, methylation, and phosphorylation. Unlike acetylation which adds a simple acetyl group, 2-hydroxyisobutyrylation involves the addition of a 2-hydroxyisobutyryl group to lysine residues on histone proteins.

This modification has unique regulatory functions in chromatin dynamics and gene expression. It's also worth noting that there are related but distinct modifications like β-hydroxybutyrylation (detected by a different antibody) that target the same lysine residue (K20) but involve different chemical groups . The specificity of these modifications suggests they may play distinct roles in cellular processes and potentially respond to different metabolic or environmental signals.

How should I design experiments to study the dynamics of 2-hydroxyisobutyryl-HIST1H2BC (K20) modification?

When studying the dynamics of 2-hydroxyisobutyryl-HIST1H2BC (K20) modification, consider the following experimental design principles:

First, establish appropriate cell or tissue models where the modification is likely to be present. Human cell lines such as HeLa, 293, A549, and K562 have been validated with this antibody. Include positive controls by treating cells with histone deacetylase inhibitors like sodium butyrate (30mM for 4 hours) to enhance histone modifications globally .

Design time-course experiments to capture the temporal dynamics of the modification in response to stimuli. Combine techniques such as Western blotting for quantification and immunofluorescence for localization. For more advanced studies, consider coupling the antibody with chromatin immunoprecipitation (ChIP) followed by sequencing to identify genomic regions associated with this modification.

Always include appropriate controls, including:

• Untreated samples as negative controls

• Isotype controls to assess non-specific binding

• Peptide competition assays to confirm antibody specificity

• Comparison with other histone marks to understand the relationship between different modifications

What are the critical considerations for sample preparation when using this antibody?

Proper sample preparation is crucial for reliable detection of 2-hydroxyisobutyryl-HIST1H2BC (K20) modification:

For histone extraction, use specialized histone extraction protocols that preserve post-translational modifications. Standard protocols often include treating cells with hypotonic lysis buffer followed by acid extraction of histones. It's essential to include protease inhibitors, phosphatase inhibitors, and critically, deacetylase inhibitors (such as sodium butyrate, trichostatin A) in your buffers to prevent loss of modifications during sample processing.

When preparing samples for Western blotting, be aware that standard reducing conditions are appropriate, but avoid excessive heat during sample denaturation as this may affect epitope recognition. For immunofluorescence, optimize fixation methods—typically, paraformaldehyde fixation (4%) for 10-15 minutes at room temperature works well, followed by permeabilization with 0.1-0.5% Triton X-100 .

Include protease and phosphatase inhibitors in all steps of sample preparation to preserve the integrity of histone modifications. Store samples at -80°C with glycerol (as in the antibody storage buffer) to prevent freeze-thaw damage if multiple experiments are planned.

How can I integrate 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody data with other epigenetic marks?

Integrating data from the 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody with other epigenetic marks requires a multi-layered approach:

First, design experiments that simultaneously examine multiple modifications. This can involve probing replicate blots with antibodies against different histone marks, performing sequential immunofluorescence studies, or conducting parallel ChIP experiments. Focus particularly on comparing 2-hydroxyisobutyrylation with related modifications such as acetylation on the same and nearby residues, as well as with the β-hydroxybutyryl modification on the same K20 site .

For bioinformatic integration, implement the following strategy:

• Perform ChIP-seq with the 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody

• Compare the genomic distribution with publicly available datasets for other histone marks

• Conduct gene ontology and pathway enrichment analyses on regions displaying co-occurrence or mutual exclusivity of marks

• Correlate the presence of marks with gene expression data to determine functional relationships

Visualization tools such as heatmaps, genome browsers, and correlation matrices can effectively display the relationships between different epigenetic marks. Advanced statistical methods including principal component analysis can help identify patterns in the distribution of multiple modifications across the genome.

What approaches should I use to investigate the role of 2-hydroxyisobutyryl-HIST1H2BC (K20) in disease mechanisms?

To investigate the role of 2-hydroxyisobutyryl-HIST1H2BC (K20) in disease mechanisms, implement a comprehensive research strategy:

Begin by comparing levels of this modification between normal and disease tissues or cell models using quantitative Western blotting with the 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody. For cancer studies, analyze a panel of cancer cell lines compared to normal counterparts. Combine this with immunohistochemistry or immunofluorescence on tissue sections to determine spatial distribution changes in the disease state .

Perform functional studies by manipulating the enzymes responsible for adding (writers) or removing (erasers) the 2-hydroxyisobutyryl mark. This can be accomplished through:

• siRNA or CRISPR-based knockdown/knockout of suspected writer/eraser enzymes

• Overexpression of these enzymes

• Small molecule inhibitors of relevant enzymatic activities

Follow with phenotypic assays to determine how alterations in this modification affect cellular processes relevant to the disease, such as proliferation, migration, or gene expression patterns. ChIP-seq analysis comparing normal and disease states can identify genomic regions where this modification is differentially distributed, revealing potential disease-associated targets .

How should I optimize Western blot protocols specifically for 2-hydroxyisobutyryl-HIST1H2BC (K20) detection?

Optimizing Western blot protocols for 2-hydroxyisobutyryl-HIST1H2BC (K20) detection requires attention to several key parameters:

Start with the recommended antibody dilution range of 1:100-1:1000, but perform a dilution series to determine the optimal concentration for your specific samples . For the primary antibody incubation, overnight at 4°C typically yields the best results for histone modification antibodies.

Sample preparation is critical—use acid extraction methods for histones and include deacetylase inhibitors like sodium butyrate in your lysis buffers. Load appropriate amounts of histone extract (typically 10-20 μg total histone protein) for optimal signal detection.

For blocking, 5% non-fat dry milk in TBST is generally effective, but 5% BSA may provide lower background for some antibody lots. Use PVDF membranes rather than nitrocellulose for better protein retention and signal strength with histone proteins.

Detection system optimization:

• For chemiluminescence: Use high-sensitivity ECL substrates

• For fluorescence: Select secondary antibodies with minimal spectral overlap if multiplexing

• Exposure time: Begin with short exposures (30 seconds) and increase gradually to avoid signal saturation

Include positive controls treated with sodium butyrate (30mM for 4 hours) to enhance the modification signal, as validated in previous studies with this antibody .

What are the best protocols for immunofluorescence staining with this antibody?

For optimal immunofluorescence staining with the 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody, follow these specialized protocols:

Cell preparation and fixation:

• Culture cells on coverslips or chamber slides to 70-80% confluence

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Wash three times with PBS

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Wash three times with PBS

Blocking and antibody incubation:

• Block with 5% normal goat serum in PBS for 1 hour at room temperature

• Incubate with 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody at the recommended dilution (1:1-1:10) in blocking buffer overnight at 4°C

• Wash five times with PBS, 5 minutes each

• Incubate with fluorescently-labeled secondary antibody (anti-rabbit) at 1:500 in blocking buffer for 1 hour at room temperature in the dark

• Wash five times with PBS, 5 minutes each

• Counterstain with DAPI (1 μg/ml) for 5 minutes

• Mount with anti-fade mounting medium

For co-staining with other histone marks, carefully select antibodies raised in different host species to allow for secondary antibody differentiation. If using multiple rabbit antibodies, consider sequential staining with direct labeling of one antibody.

Include appropriate controls:

• Secondary antibody only (no primary) to assess background

• Isotype control antibody to determine non-specific binding

• Peptide competition to confirm specificity

• Positive control (sodium butyrate-treated cells)

How do I interpret conflicting results between 2-hydroxyisobutyryl-HIST1H2BC (K20) and other histone marks?

When faced with conflicting results between 2-hydroxyisobutyryl-HIST1H2BC (K20) and other histone marks, consider several potential explanations and verification approaches:

First, understand that different histone modifications may have antagonistic relationships. The presence of one modification can inhibit or enhance the deposition of another at the same or nearby residues. For instance, 2-hydroxyisobutyrylation at K20 may compete with acetylation or β-hydroxybutyrylation at the same residue .

To systematically address conflicting results:

• Verify antibody specificity for each modification using peptide competition assays

• Confirm results with alternative antibodies targeting the same modifications

• Use mass spectrometry-based approaches as an antibody-independent method to validate the presence and stoichiometry of modifications

• Conduct temporal analyses to determine if modifications appear sequentially rather than simultaneously

• Investigate the enzymes responsible for each modification to understand potential regulatory cross-talk

Different cell types, developmental stages, or environmental conditions may show distinct modification patterns. Standardize experimental conditions and perform biological replicates to ensure reproducibility. Sometimes apparent conflicts may reveal important biological insights about the dynamic nature of chromatin regulation.

What are the common pitfalls in using this antibody and how can they be avoided?

Several common pitfalls can affect experiments with the 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody:

• Loss of modifications during sample preparation

  • Pitfall: Histone modifications can be lost during extraction and processing

  • Solution: Always include deacetylase inhibitors (sodium butyrate, TSA, etc.) in all buffers, and minimize processing time

• Non-specific binding

  • Pitfall: Polyclonal antibodies may show some cross-reactivity

  • Solution: Use longer blocking times (2 hours), higher BSA concentrations in blocking buffers (3-5%), and thoroughly validate specificity with peptide competition assays

• Suboptimal signal in Western blots

  • Pitfall: Weak or absent signal despite proper technique

  • Solution: Increase protein loading (15-20 μg of histone extract), optimize antibody concentration, and extend exposure times. Consider enhancing the modification with sodium butyrate treatment (30mM for 4 hours) as a positive control

• High background in immunofluorescence

  • Pitfall: Non-specific nuclear staining making specific signal difficult to distinguish

  • Solution: Increase washing steps (5-6 washes of 10 minutes each), use more dilute antibody concentration, and optimize fixation conditions

• Batch-to-batch variability

  • Pitfall: Different lots may have slightly different performance characteristics

  • Solution: Test each new lot side-by-side with previous successful lot, and maintain consistent experimental conditions

• Storage and handling issues

  • Pitfall: Repeated freeze-thaw cycles can diminish antibody performance

  • Solution: Aliquot the antibody upon receipt and store at -20°C or -80°C. Follow manufacturer guidelines for storage in 50% glycerol with preservatives

How can I integrate 2-hydroxyisobutyryl-HIST1H2BC (K20) analysis with metabolomic studies?

Integrating 2-hydroxyisobutyryl-HIST1H2BC (K20) analysis with metabolomics creates an innovative approach to understanding how cellular metabolism impacts epigenetic regulation:

Since histone 2-hydroxyisobutyrylation utilizes 2-hydroxyisobutyryl-CoA as a substrate, which is derived from cellular metabolic pathways, changes in cellular metabolism can directly impact this modification. Design experiments that manipulate relevant metabolic pathways while monitoring changes in 2-hydroxyisobutyryl-HIST1H2BC (K20) levels.

Experimental approach:

• Manipulate cellular metabolism through nutrient restriction, supplementation, or metabolic inhibitors

• Measure global levels of 2-hydroxyisobutyryl-HIST1H2BC (K20) by Western blotting

• Perform ChIP-seq with the antibody to identify genomic regions affected by metabolic changes

• In parallel, conduct targeted metabolomics to measure levels of relevant metabolites, particularly those involved in 2-hydroxyisobutyryl-CoA production

• Correlate metabolite levels with changes in the histone modification pattern

• Integrate with transcriptomics to identify genes whose expression correlates with both metabolite levels and 2-hydroxyisobutyryl-HIST1H2BC (K20) occupancy

This integrated approach can reveal how specific metabolic states influence gene expression through chromatin modifications, providing insights into diseases with both metabolic and epigenetic components, such as cancer, diabetes, and neurological disorders .

What are the most promising techniques for genome-wide mapping of 2-hydroxyisobutyryl-HIST1H2BC (K20)?

For genome-wide mapping of 2-hydroxyisobutyryl-HIST1H2BC (K20), several cutting-edge techniques offer distinct advantages:

Standard ChIP-seq
This established technique uses the 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody to immunoprecipitate modified chromatin, followed by next-generation sequencing. For optimal results, use 5-10 μg of antibody per 10-30 μg of chromatin, and include appropriate controls such as input DNA and IgG immunoprecipitation.

CUT&RUN (Cleavage Under Targets and Release Using Nuclease)
This technique offers improved signal-to-noise ratio compared to conventional ChIP-seq:

• Immobilize cells on Concanavalin A-coated magnetic beads

• Permeabilize cells and incubate with 2-hydroxyisobutyryl-HIST1H2BC (K20) antibody

• Add protein A-MNase fusion protein, which binds to the antibody

• Activate MNase with calcium to cleave DNA around the modification site

• Release and sequence the cleaved fragments

CUT&Tag (Cleavage Under Targets and Tagmentation)
This method improves efficiency through direct tagmentation:

• Follow similar steps as CUT&RUN, but use protein A-Tn5 transposase fusion

• The transposase simultaneously cleaves and adds sequencing adapters

• This requires less starting material and fewer amplification cycles

Single-cell approaches
For heterogeneity studies, adapt CUT&Tag for single-cell applications:

• Perform CUT&Tag on a cell suspension

• Encapsulate cells in droplets or use well-based methods

• Add cell-specific barcodes during library preparation

• This reveals cell-to-cell variation in 2-hydroxyisobutyryl-HIST1H2BC (K20) distribution

Bioinformatic analysis should include specialized peak-calling algorithms optimized for histone modifications (broad peaks) rather than transcription factors (narrow peaks). Integrate with existing datasets on gene expression and other histone marks to contextualize the specific role of 2-hydroxyisobutyryl-HIST1H2BC (K20) in gene regulation .

How does 2-hydroxyisobutyryl-HIST1H2BC (K20) modification compare with 2-hydroxyisobutyrylation at other lysine residues?

2-hydroxyisobutyrylation occurs at multiple lysine residues in histones, with distinct distributional patterns and likely different functional implications:

The 2-hydroxyisobutyryl modification at K20 of HIST1H2BC appears to have specific characteristics compared to the same modification at other residues such as K12 or K120 on the same histone. While all these modifications involve the addition of a 2-hydroxyisobutyryl group, their genomic distributions and functional roles may differ significantly .

To systematically compare these modifications:

• Perform sequential ChIP (re-ChIP) experiments to determine if these modifications co-occur at the same genomic loci or exist mutually exclusively

• Analyze the enzymes responsible for adding and removing these modifications at different sites

• Correlate each modification site with specific transcriptional states or chromatin features

• Compare cellular conditions that enhance or diminish each specific modification

Understanding these site-specific differences can provide insights into the "histone code" and how different positions of the same modification may convey distinct regulatory information .

What are the key differences between 2-hydroxyisobutyrylation and β-hydroxybutyrylation at HIST1H2BC (K20)?

2-hydroxyisobutyrylation and β-hydroxybutyrylation represent distinct chemical modifications that can occur at the same K20 residue of HIST1H2BC but likely have different functional implications:

These two modifications, while chemically similar, likely respond to distinct metabolic conditions within the cell. β-hydroxybutyrylation is thought to increase during fasting, ketogenic diets, or diabetic ketoacidosis when β-hydroxybutyrate levels rise. In contrast, 2-hydroxyisobutyrylation may respond to different metabolic signals related to branched-chain amino acid metabolism .

To experimentally distinguish between these modifications:

• Use the specific antibodies for each modification in parallel experiments

• Manipulate cellular metabolism to enhance each modification selectively

  • Ketogenic conditions for β-hydroxybutyrylation

  • Branched-chain amino acid supplementation for 2-hydroxyisobutyrylation

• Perform mass spectrometry analyses to confirm the chemical identity of each modification

• Compare genomic distributions through ChIP-seq with each specific antibody

Understanding the differential regulation and function of these similar but distinct modifications provides insight into how cells use chromatin to integrate diverse metabolic signals .