β-hydroxybutyryl-HIST1H1D (K106) Antibody

What is β-hydroxybutyryl-HIST1H1D (K106) and why is it important in epigenetic research?

β-hydroxybutyryl-HIST1H1D (K106) refers to a specific post-translational modification where lysine 106 of Histone H1.3 (also known as HIST1H1D) is modified by the addition of a β-hydroxybutyryl group. This modification is part of a broader class of histone marks that regulate chromatin structure and gene expression. The importance of this modification lies in its role within the complex landscape of epigenetic regulation. Histone H1.3 functions as a linker histone that binds to DNA between nucleosomes, contributing to higher-order chromatin structure and gene regulation . The β-hydroxybutyrylation of this specific lysine residue likely modulates these functions, potentially affecting accessibility of transcription factors and other regulatory proteins to DNA.

The antibody against this modification enables researchers to specifically detect and study this epigenetic mark in various experimental contexts, from cell culture to tissue samples. Understanding the patterns and dynamics of this modification contributes to our knowledge of how metabolic signals may be integrated into the epigenome, as β-hydroxybutyrylation is linked to cellular metabolism through β-hydroxybutyrate, a ketone body produced during fasting or ketogenic diets .

How does β-hydroxybutyrylation differ from other histone modifications such as acetylation or methylation?

β-hydroxybutyrylation represents a distinct class of histone post-translational modification that differs from more extensively studied modifications like acetylation and methylation in several important ways:

• Chemical Structure: β-hydroxybutyrylation involves the addition of a four-carbon β-hydroxybutyryl group (C₄H₇O₃) to lysine residues, which is larger than the acetyl group (C₂H₃O) in acetylation but functionally different from methyl groups added during methylation.

• Metabolic Connection: Unlike acetylation, which is linked to acetyl-CoA levels, β-hydroxybutyrylation is associated with β-hydroxybutyrate metabolism, connecting this modification to ketone body production during fasting, caloric restriction, or ketogenic diets .

• Functional Impact: While both acetylation and β-hydroxybutyrylation neutralize the positive charge of lysine residues, potentially loosening histone-DNA interactions, β-hydroxybutyrylation may recruit a different set of reader proteins that specifically recognize this modification, leading to distinct downstream effects on gene expression .

• Regulatory Enzymes: The enzymes that catalyze the addition and removal of β-hydroxybutyryl groups likely differ from the histone acetyltransferases (HATs) and histone deacetylases (HDACs) that regulate acetylation, though some overlap in enzyme function may exist.

Understanding these differences is crucial for correctly interpreting experimental results and designing appropriate controls when using the β-hydroxybutyryl-HIST1H1D (K106) antibody in research studies.

What are the validated applications for β-hydroxybutyryl-HIST1H1D (K106) Antibody?

The β-hydroxybutyryl-HIST1H1D (K106) antibody has been validated for several experimental applications, each requiring specific optimization:

These applications enable researchers to investigate the presence, abundance, and localization of β-hydroxybutyryl-HIST1H1D (K106) in various biological contexts . The antibody binds specifically to the peptide sequence surrounding the β-hydroxybutyrylated lysine 106 residue of Human Histone H1.3, allowing for reliable detection of this modification.

For Western blot applications, the antibody can detect bands around 23 kDa, corresponding to the expected molecular weight of Histone H1.3. Optimal results may require pre-treatment of samples with sodium butyrate to increase the levels of β-hydroxybutyrylation, similar to protocols used for the K75 site .

How can I optimize ChIP protocols for use with β-hydroxybutyryl-HIST1H1D (K106) Antibody?

While Chromatin Immunoprecipitation (ChIP) is not listed among the validated applications for this specific antibody in the provided information, researchers can adapt standard ChIP protocols with the following optimization strategies:

A successful ChIP protocol will likely require empirical optimization for each cell type and experimental condition being studied.

How do I verify the specificity of β-hydroxybutyryl-HIST1H1D (K106) Antibody?

Ensuring antibody specificity is critical for generating reliable research data. For β-hydroxybutyryl-HIST1H1D (K106) antibody, consider implementing the following validation approaches:

• Peptide Competition Assay: Pre-incubate the antibody with excess modified peptide (containing β-hydroxybutyrylated K106) before application in your experiment. A specific antibody will show greatly reduced or eliminated signal after peptide blocking.

• Modified vs. Unmodified Peptide Arrays: Test the antibody against arrays containing both the β-hydroxybutyrylated K106 peptide and unmodified versions to confirm specific binding only to the modified form.

• Knockdown/Knockout Controls: Where possible, use cells with reduced expression of HIST1H1D or cells treated with histone deacetylase inhibitors that may affect β-hydroxybutyrylation levels.

• Cross-reactivity Testing: Assess potential cross-reactivity with other β-hydroxybutyrylated histones or with other modifications at the same site (e.g., acetylation, methylation) using dot blots or Western blots.

• Mass Spectrometry Validation: For the most rigorous validation, use immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down HIST1H1D with β-hydroxybutyrylation specifically at K106.

• Positive Control Samples: Include samples known to have high levels of the modification, such as cells treated with sodium butyrate, which has been shown to increase β-hydroxybutyrylation levels .

Validation is especially important when studying histone modifications due to the potential for cross-reactivity with similar modifications or the same modification at different lysine residues.

What factors influence the reliability of results when using this antibody?

Several factors can significantly impact the reliability of results obtained with β-hydroxybutyryl-HIST1H1D (K106) antibody:

• Antibody Quality and Storage: Antibody degradation due to improper storage or handling can reduce specificity and sensitivity. Store according to manufacturer recommendations, typically at -20°C or -80°C in small aliquots to avoid freeze-thaw cycles .

• Sample Preparation: Improper fixation, extraction, or processing can affect epitope accessibility. For cell preparations, use freshly prepared formaldehyde for fixation and optimize fixation times for your specific cell type .

• Experimental Conditions:

  • pH and salt concentration of buffers

  • Incubation temperature and duration

  • Blocking reagent selection and concentration

  • Secondary antibody selection and optimization

• Biological Variables:

  • Cell cycle stage (histone modifications often vary with cell cycle)

  • Metabolic state of cells (particularly relevant for β-hydroxybutyrylation)

  • Stress conditions or treatments that may alter global histone modification patterns

• Technical Variables:

  • Batch-to-batch variation in antibody production

  • Variation in equipment calibration or protocol execution

• Controls and Normalization:

  • Inclusion of appropriate positive and negative controls

  • Consistent normalization strategies across experiments

To enhance reliability, it is recommended to validate the antibody under your specific experimental conditions before proceeding with full-scale experiments .

How can I troubleshoot weak or nonspecific signals when using β-hydroxybutyryl-HIST1H1D (K106) Antibody?

When encountering weak or nonspecific signals, consider the following troubleshooting approaches:

For Weak Signals:

• Antibody Concentration: Increase antibody concentration within the recommended range (1:100-1:1000 for WB or 1:10-1:100 for ICC) .

• Epitope Retrieval: For fixed samples, optimize antigen retrieval methods (heat-induced or enzyme-based).

• Signal Amplification: Consider using more sensitive detection systems such as tyramide signal amplification (TSA).

• Sample Amount: Increase the amount of protein loaded for Western blots.

• Exposure Time: Extend exposure time when acquiring images, while monitoring background.

• Metabolic Stimulation: Treat cells with sodium butyrate (30 mM for 4 hours) to increase β-hydroxybutyrylation levels before analysis .

For Nonspecific Signals:

• Blocking Optimization: Test different blocking agents (BSA, milk, normal serum) and concentrations.

• Washing Stringency: Increase the number or duration of washes, or adjust salt concentration in wash buffers.

• Antibody Dilution: Increase antibody dilution to reduce nonspecific binding.

• Pre-adsorption: Pre-adsorb the antibody with cell/tissue lysates from species different from your sample.

• Secondary Antibody: Ensure secondary antibody is appropriate for your primary antibody species and isotype (IgG) .

• Reducing Agents: Include reducing agents in buffer to prevent disulfide bond formation in the antibody.

For both issues, isolating nuclei before protein extraction can enrich for histone proteins and improve signal-to-noise ratio in most applications.

How can I integrate β-hydroxybutyryl-HIST1H1D (K106) data with other epigenetic marks?

Integrating data from multiple epigenetic marks provides a more comprehensive understanding of chromatin regulation. Consider these methodological approaches:

• Sequential Chromatin Immunoprecipitation (Re-ChIP): This technique involves performing successive rounds of immunoprecipitation with different antibodies to identify genomic regions containing both marks, potentially revealing functional relationships between β-hydroxybutyryl-HIST1H1D (K106) and other epigenetic modifications.

• Multiplexed Immunofluorescence: For tissue or cell imaging, use different fluorophores conjugated to secondary antibodies against various histone modification antibodies. Ensure appropriate controls for antibody cross-reactivity and spectral overlap.

• Mass Spectrometry-Based Approaches: Bottom-up proteomics can identify co-occurring histone modifications on the same histone tail or within the same nucleosome, providing insight into modification patterns.

• Bioinformatic Integration:

  • For ChIP-seq data, use tools like deepTools, ChromHMM, or GIGGLE to integrate multiple datasets

  • Apply machine learning approaches to identify patterns across different marks

  • Use genome browsers to visualize the co-occurrence of different marks at specific loci

• Correlation Analysis: Calculate correlation coefficients between β-hydroxybutyryl-HIST1H1D (K106) and other histone marks across genomic regions to identify potential functional relationships.

• Experimental Manipulation: Perturb one modification using small molecule inhibitors or genetic approaches, then measure effects on other modifications to establish causal relationships .

When designing such integrated analyses, consider the biological question carefully and ensure that statistical methods account for the complex relationships between different epigenetic marks.

How is β-hydroxybutyryl-HIST1H1D (K106) being investigated in disease contexts?

While specific research on β-hydroxybutyryl-HIST1H1D (K106) in disease contexts is limited in the provided information, emerging research on histone β-hydroxybutyrylation and HIST1H1D provides insights into potential disease relevance:

• Cancer Research: Dysregulation of Histone H1 variants, including HIST1H1D, has been implicated in various cancers. Altered expression patterns of HIST1H1D may contribute to aberrant chromatin structure and gene expression in cancer cells . The specific role of β-hydroxybutyrylation at K106 in cancer progression or suppression represents an emerging area of investigation.

• Neurological Disorders: HIST1H1D dysregulation has been linked to neurological disorders . Given the connection between β-hydroxybutyrylation and metabolism, this modification may be particularly relevant in neurodegenerative diseases with metabolic components, such as Alzheimer's disease.

• Metabolic Diseases: β-hydroxybutyrylation serves as a link between cellular metabolism and epigenetic regulation. Research is emerging on how this modification may contribute to metabolic disorders such as diabetes and obesity, though specific studies on the K106 site are still developing.

• Inflammatory Conditions: Histone modifications play crucial roles in regulating inflammatory responses. The role of β-hydroxybutyryl-HIST1H1D (K106) in modulating inflammatory gene expression represents a potential area for therapeutic intervention.

• Aging-Related Disorders: Changes in histone modification patterns occur during aging. Understanding how β-hydroxybutyryl-HIST1H1D (K106) changes with age could provide insights into age-related diseases and potential interventions.

For researchers investigating β-hydroxybutyryl-HIST1H1D (K106) in disease contexts, it is advisable to incorporate tissue microarrays or patient sample cohorts with appropriate controls and correlate findings with clinical outcomes to establish disease relevance.

What emerging technologies are enhancing research with β-hydroxybutyryl-HIST1H1D (K106) Antibody?

Several cutting-edge technologies are expanding the capabilities of research using histone modification antibodies like β-hydroxybutyryl-HIST1H1D (K106):

• Single-Cell Epigenomics: Technologies like CUT&Tag, CUT&RUN, and single-cell ChIP-seq allow for profiling of histone modifications at single-cell resolution, revealing cell-to-cell heterogeneity in β-hydroxybutyrylation patterns that may be masked in bulk analyses.

• Spatial Epigenomics: Methods combining immunofluorescence with spatial transcriptomics enable visualization of β-hydroxybutyryl-HIST1H1D (K106) distribution within the context of tissue architecture, providing insights into its role in tissue organization and function.

• CRISPR Epigenome Editing: CRISPR-based systems coupled with histone modifying enzymes allow for site-specific manipulation of β-hydroxybutyrylation, enabling causal studies of this modification's function at specific genomic loci.

• Proximity Ligation Assays: These techniques can detect interactions between β-hydroxybutyryl-HIST1H1D (K106) and other proteins or histone marks in situ, revealing functional protein complexes associated with this modification.

• Advanced Mass Spectrometry: Improvements in sensitivity and throughput of mass spectrometry enable comprehensive profiling of histone modifications, including identification of novel sites and combinations of modifications .

• Synthetic Histone Technology: Production of designer histones with specific modifications allows for mechanistic studies on how β-hydroxybutyrylation affects nucleosome structure and function.

• Microfluidic Approaches: Miniaturized assay platforms enable high-throughput screening of factors affecting β-hydroxybutyrylation levels or the development of more sensitive detection methods requiring minimal sample input.

These technologies offer researchers powerful tools to investigate the biological functions and disease relevance of β-hydroxybutyryl-HIST1H1D (K106) with unprecedented resolution and specificity .