β-hydroxybutyryl-HIST1H3A (K122) Antibody

What is β-hydroxybutyryl-HIST1H3A (K122) antibody and what biological function does it detect?

β-hydroxybutyryl-HIST1H3A (K122) antibody specifically recognizes the β-hydroxybutyrylation post-translational modification at lysine 122 of histone H3.1 protein. This antibody detects a critical epigenetic modification that plays a role in transcriptional regulation and chromatin structure modulation . Histone H3.1 is a core component of nucleosomes that wrap and compact DNA into chromatin, thereby limiting DNA accessibility to cellular machineries that require DNA as a template . The β-hydroxybutyrylation modification at K122 is part of the complex "histone code" that regulates DNA accessibility, affecting transcription regulation, DNA repair, DNA replication, and chromosomal stability .

What are the validated research applications for this antibody?

The β-hydroxybutyryl-HIST1H3A (K122) antibody has been validated for multiple research applications including:

The antibody has been specifically tested and validated in human cell lysates, including 293 and HepG2 cell lines treated with sodium butyrate . For immunocytochemistry applications, successful staining has been demonstrated in HeLa cells treated with 50mM sodium 3-hydroxybutyrate for 72 hours .

What is the species reactivity profile of this antibody?

The β-hydroxybutyryl-HIST1H3A (K122) antibody primarily reacts with human samples . Some suppliers also report reactivity with rat samples . The antibody was raised using a peptide sequence derived from the region surrounding the β-hydroxybutyryl-Lys (122) site of human Histone H3.1 (UniProt accession P68431) . No cross-reactivity with mouse or other species has been specifically validated in the available literature. Researchers working with non-human models should perform validation tests before proceeding with large-scale experiments.

What are the optimal storage and handling conditions for this antibody?

For optimal performance and longevity, the β-hydroxybutyryl-HIST1H3A (K122) antibody should be:

• Stored at -20°C for long-term preservation

• Aliquoted upon receipt to minimize freeze-thaw cycles

• Avoided repeated freeze/thaw cycles which can diminish antibody activity

The antibody is typically supplied in a liquid format with a buffer composition of 0.01M PBS (pH 7.4), 50% glycerol, and 0.03% Proclin-300 as a preservative . This formulation helps maintain antibody stability during storage and prevents microbial contamination.

How should I design positive and negative controls for β-hydroxybutyryl-HIST1H3A (K122) antibody experiments?

Positive Controls:

• Treatment of human cell lines with sodium butyrate (30mM for 4 hours) has been demonstrated to induce β-hydroxybutyrylation of histones, making these samples suitable positive controls for Western blot applications .

• Alternatively, treatment with sodium 3-hydroxybutyrate (50mM for 72 hours) in HeLa cells provides a robust positive control for both Western blot and immunocytochemistry applications .

Negative Controls:

• Primary antibody omission - replace the primary antibody with the same concentration of non-immune rabbit IgG

• Peptide competition assay - pre-incubate the antibody with excess immunizing peptide to demonstrate binding specificity

• CRISPR/Cas9 knockout or knockdown of HIST1H3A in cell lines can serve as genetic negative controls

When designing control experiments, it is essential to maintain consistent sample preparation, incubation times, and detection methods across all experimental and control conditions.

What are the recommended blocking and incubation conditions for Western blot and ICC applications?

Western Blot Recommendations:

• Transfer proteins to PVDF or nitrocellulose membrane

• Block with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature

• Incubate with primary antibody (β-hydroxybutyryl-HIST1H3A K122) at 1:100-1:1000 dilution in blocking buffer overnight at 4°C

• Wash 3-4 times with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary anti-rabbit antibody at 1:5000-1:50000 dilution for 1 hour at room temperature

• Develop using ECL or similar chemiluminescence system

Immunocytochemistry Recommendations:

• Fix cells in 4% formaldehyde

• Permeabilize using 0.2% Triton X-100

• Block with 10% normal goat serum for 30 minutes at room temperature

• Incubate with primary antibody at 1:10-1:100 dilution in 1% BSA overnight at 4°C

• Detect using biotinylated secondary antibody and visualize with HRP-conjugated reagents or fluorescent detection systems

These protocols have been validated with human cell lines and should be optimized for specific experimental conditions.

What factors might affect epitope accessibility when using this antibody?

Several factors can influence the accessibility of the β-hydroxybutyryl modification at lysine 122 of histone H3.1:

• Fixation conditions: Overfixation with formaldehyde can mask epitopes. For ICC applications, optimize fixation time (typically 10-15 minutes with 4% formaldehyde) .

• Chromatin state: The nucleosome structure may limit accessibility to histone modifications. Consider using epitope retrieval methods for tissue sections or highly condensed chromatin.

• Neighboring modifications: Adjacent histone modifications can interfere with antibody binding. Characterize the modification landscape of your experimental system.

• Protein-protein interactions: Transcription factors or chromatin remodelers bound to the region may block antibody access.

• Protein extraction methods: For Western blot applications, ensure complete histone extraction using acidic extraction methods rather than standard RIPA buffer protocols.

To enhance epitope accessibility, incorporating antigen retrieval steps or using specialized extraction buffers for histones can significantly improve detection sensitivity.

How can I use the β-hydroxybutyryl-HIST1H3A (K122) antibody in ChIP or ChIP-seq experiments?

While the antibody product information doesn't specifically list ChIP (Chromatin Immunoprecipitation) as a validated application, researchers can adapt this antibody for ChIP experiments based on established protocols for other histone modification antibodies. Consider the following methodology:

• Crosslinking: Treat cells with 1% formaldehyde for 10 minutes at room temperature to crosslink DNA-protein complexes.

• Chromatin preparation: Lyse cells, sonicate chromatin to achieve 200-500bp fragments, and confirm fragmentation quality by agarose gel electrophoresis.

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate 2-5μg antibody with chromatin overnight at 4°C

  • Add protein A/G beads and incubate for 2-4 hours

  • Wash with increasing stringency buffers

• Elution and analysis:

  • Elute DNA-protein complexes and reverse crosslinks

  • Purify DNA for qPCR or next-generation sequencing

• Controls: Include IgG control, input chromatin control, and positive control using an established histone mark antibody (H3K4me3 or H3K27ac).

For ChIP-seq applications, ensure high-quality antibody specificity by validating with peptide competition assays or using cells treated with sodium 3-hydroxybutyrate to enhance the signal-to-noise ratio.

What are the key considerations for multiplex immunofluorescence using β-hydroxybutyryl-HIST1H3A (K122) antibody?

When designing multiplex immunofluorescence experiments with this antibody, consider:

• Antibody compatibility: The β-hydroxybutyryl-HIST1H3A (K122) antibody is rabbit-derived , requiring careful selection of co-staining antibodies from different host species (mouse, goat, or chicken) to avoid cross-reactivity.

• Sequential staining protocol:

  • Begin with the lowest abundance target (often β-hydroxybutyryl-HIST1H3A)

  • Use tyramide signal amplification for weak signals

  • Employ heat-mediated stripping between rounds of staining

• Spectral considerations: Choose fluorophores with minimal spectral overlap and account for tissue autofluorescence, particularly in FFPE samples.

• Controls for multiplex staining:

  • Single antibody controls to establish signal specificity

  • Secondary-only controls to assess background

  • Absorption controls with immunizing peptide

• Image acquisition and analysis: Use spectral unmixing algorithms for closely overlapping fluorophores and conduct pixel-by-pixel colocalization analysis when studying relationships between β-hydroxybutyrylation and other histone marks.

A robust multiplex panel might include β-hydroxybutyryl-HIST1H3A (K122) alongside markers for transcriptional activity (RNA Pol II), other histone modifications (H3K27ac), and cell-type specific markers relevant to your research question.

How does metabolic state influence β-hydroxybutyrylation patterns detectable by this antibody?

β-hydroxybutyrylation of histones represents a direct link between cellular metabolism and epigenetic regulation . Several metabolic conditions can influence the patterns detectable by the β-hydroxybutyryl-HIST1H3A (K122) antibody:

• Ketogenic states: During fasting, starvation, or ketogenic diet, elevated β-hydroxybutyrate levels in the blood and tissues can increase histone β-hydroxybutyrylation. Treat cells with 3-hydroxybutyrate (30-50mM) to mimic this condition experimentally .

• Diabetes and insulin resistance: Altered metabolic states in diabetes may affect histone β-hydroxybutyrylation patterns. Research models of insulin resistance could show different modification profiles.

• Mitochondrial dysfunction: Conditions that impact mitochondrial fatty acid oxidation may alter ketone body production and subsequent histone modification.

• Circadian rhythm: Metabolic fluctuations throughout the day can influence histone modifications, suggesting time-course experiments may reveal temporal dynamics of β-hydroxybutyrylation.

When designing experiments to study metabolic influences on histone β-hydroxybutyrylation, researchers should consider:

• Careful documentation of nutritional status of cell cultures or animal models

• Measurement of β-hydroxybutyrate levels in experimental systems

• Parallel analysis of key metabolic enzymes involved in ketone body metabolism

• Integration with other epigenetic marks to establish a comprehensive modification landscape

What are common issues in Western blot detection using β-hydroxybutyryl-HIST1H3A (K122) antibody and how can they be resolved?

The expected molecular weight for histone H3.1 is approximately 16 kDa as observed in validated Western blots . When troubleshooting, always run appropriate controls including acid-extracted histones from cells treated with sodium butyrate or β-hydroxybutyrate as positive controls.

How can I quantitatively analyze β-hydroxybutyrylation levels in different experimental conditions?

For quantitative analysis of β-hydroxybutyrylation levels across different experimental conditions, consider these methodological approaches:

• Western blot densitometry:

  • Normalize β-hydroxybutyryl-HIST1H3A (K122) signal to total H3 loading control

  • Use standard curve of recombinant or synthetic β-hydroxybutyrylated peptides

  • Employ technical triplicates and biological replicates

• ELISA-based quantification:

  • Develop a sandwich ELISA using anti-H3 capture antibody and β-hydroxybutyryl-HIST1H3A (K122) detection antibody

  • Create standard curves using synthetic modified peptides

  • Recommended antibody dilution for ELISA: 1:2000-1:10000

• Mass spectrometry approach:

  • Use antibody for immunoprecipitation followed by mass spectrometry

  • Employ parallel reaction monitoring (PRM) for targeted quantification

  • Incorporate isotopically labeled peptide standards for absolute quantification

• Imaging-based quantification:

  • For ICC/IF analysis, use automated image analysis software

  • Measure nuclear intensity of β-hydroxybutyryl-HIST1H3A staining

  • Normalize to DAPI or total H3 staining

  • Include at least 100 cells per condition for statistical robustness

When comparing β-hydroxybutyrylation levels between experimental conditions, ensure:

• Consistent sample preparation

• Simultaneous processing of all samples

• Inclusion of technical and biological replicates

• Appropriate statistical analysis (e.g., t-test, ANOVA)

How can I differentiate between β-hydroxybutyrylation and other acyl modifications in experimental systems?

Distinguishing β-hydroxybutyrylation from other histone acyl modifications requires careful experimental design and controls:

• Antibody specificity validation:

  • Perform peptide competition assays with β-hydroxybutyrylated, acetylated, and butyrylated peptides

  • Use dot blot analysis with modified peptide arrays containing various acyl modifications

  • Consider Western blot analysis with recombinant histones containing defined modifications

• Mass spectrometry differentiation:

  • β-hydroxybutyrylation adds a mass of 86.05 Da, distinguishable from acetylation (42.01 Da) and butyrylation (70.04 Da)

  • High-resolution MS can differentiate these modifications based on mass differences

  • Use fragmentation patterns unique to each modification for validation

• Metabolic manipulation:

  • Treat cells with specific precursors for each modification:

    • Sodium butyrate for butyrylation

    • Sodium acetate for acetylation

    • 3-hydroxybutyrate for β-hydroxybutyrylation

  • Compare modification patterns to identify specific targets

• Enzymatic sensitivity:

  • Different deacylases show specificity for certain modifications

  • Use selective HDAC inhibitors to distinguish sensitivity profiles

  • Monitor effects of sirtuin activators/inhibitors on modification levels

For comprehensive analysis, consider using multiple detection methods in parallel, as each approach has inherent limitations. Integration of biochemical, immunological, and mass spectrometry techniques provides the most robust differentiation between these closely related histone modifications.

What are the emerging research areas employing β-hydroxybutyryl-HIST1H3A (K122) antibody?

The β-hydroxybutyryl-HIST1H3A (K122) antibody is increasingly being utilized in several cutting-edge research areas:

• Metabolic epigenetics: Investigating how metabolic states (fasting, ketosis, diabetes) influence gene expression through histone β-hydroxybutyrylation .

• Neurodegenerative disorders: Exploring the neuroprotective effects of ketone bodies through histone modifications in models of Alzheimer's, Parkinson's, and other neurodegenerative conditions.

• Cancer metabolism: Studying how altered metabolism in cancer cells impacts epigenetic landscapes, potentially revealing novel therapeutic vulnerabilities.

• Inflammatory conditions: Investigating the role of β-hydroxybutyrylation in regulating inflammatory gene expression, particularly in conditions where the ketogenic diet shows therapeutic potential.

• Aging research: Examining how β-hydroxybutyrylation patterns change with age and whether interventions like caloric restriction or intermittent fasting impact these modifications.

• Exercise physiology: Analyzing how different exercise protocols alter ketone body levels and subsequent histone modifications relevant to muscle adaptation and performance.

These emerging areas highlight the importance of specific detection tools like the β-hydroxybutyryl-HIST1H3A (K122) antibody in connecting metabolic status to epigenetic regulation mechanisms.

How can ChIP-seq with β-hydroxybutyryl-HIST1H3A (K122) antibody provide insights into gene regulation?

ChIP-seq using the β-hydroxybutyryl-HIST1H3A (K122) antibody can provide comprehensive insights into gene regulation mechanisms through:

• Genome-wide occupancy mapping:

  • Identify genes and regulatory elements associated with β-hydroxybutyrylation at H3K122

  • Compare with known transcriptionally active regions to establish functional correlations

  • Integrate with RNA-seq data to directly link modification to gene expression changes

• Metabolic state comparisons:

  • Map β-hydroxybutyrylation patterns in different metabolic conditions (fed vs. fasted, normal vs. ketogenic diet)

  • Identify differentially modified regions that respond to metabolic shifts

  • Discover metabolically responsive gene networks

• Integration with other histone modifications:

  • Combine with ChIP-seq data for other marks (H3K27ac, H3K4me3, etc.)

  • Develop comprehensive epigenetic signatures associated with specific cellular states

  • Identify unique and overlapping target genes between β-hydroxybutyrylation and other modifications

• Transcription factor co-occurrence:

  • Analyze co-occurrence with transcription factor binding sites

  • Identify potential readers and erasers of the modification

  • Establish mechanistic links between metabolism and transcriptional machinery

When designing ChIP-seq experiments with this antibody, researchers should:

• Ensure high antibody specificity with appropriate controls

• Use sufficient sequencing depth (>20 million uniquely mapped reads)

• Include input controls and IgG controls

• Consider biological replicates for robust peak calling

What are the recommended approaches for studying the dynamics of β-hydroxybutyrylation in living systems?

To study the dynamics of β-hydroxybutyrylation in living systems, researchers can employ several complementary approaches:

• Time-course experiments:

  • Monitor β-hydroxybutyrylation changes during metabolic transitions (glucose to ketone metabolism)

  • Collect samples at multiple timepoints (0h, 2h, 4h, 8h, 24h, etc.)

  • Correlate with metabolite measurements (β-hydroxybutyrate levels in media or serum)

• In vivo models:

  • Use diet manipulation (standard diet vs. ketogenic diet)

  • Employ fasting/feeding cycles to naturally modulate ketone levels

  • Consider genetic models with altered ketone metabolism

• Live-cell imaging techniques:

  • Develop fluorescent biosensors for β-hydroxybutyrylation

  • Use FRET-based reporters to monitor modification dynamics

  • Apply photobleaching techniques to assess turnover rates

• Metabolic isotope tracing:

  • Use stable isotope-labeled β-hydroxybutyrate (13C or D)

  • Track incorporation into histone modifications using mass spectrometry

  • Calculate rates of turnover for specific modification sites

• Enzyme manipulation studies:

  • Overexpress or inhibit putative "writers" of β-hydroxybutyrylation

  • Modulate "erasers" to assess removal dynamics

  • Employ CRISPR/Cas9 to create site-specific histone mutants (K122R)

When designing these experiments, researchers should:

• Carefully control nutritional conditions

• Monitor relevant metabolite levels throughout the experiment

• Consider cell-type specific responses

• Integrate multiple analytical techniques for comprehensive understanding

What are the current limitations of β-hydroxybutyryl-HIST1H3A (K122) antibody-based research?

Despite its utility, several limitations exist in current β-hydroxybutyryl-HIST1H3A (K122) antibody-based research:

• Specificity challenges: Absolute specificity between closely related acyl modifications remains difficult to fully validate without extensive controls.

• Limited species validation: Current antibodies are primarily validated for human samples with limited cross-species validation data available .

• Technical variability: Inconsistencies between antibody lots and preparation methods can complicate cross-study comparisons.

• Resolution limitations: Antibody-based approaches cannot distinguish between specific histone variants with identical modification sites.

• Context dependency: The antibody may have different accessibility to the epitope depending on neighboring modifications or protein-protein interactions.

• Quantification challenges: Western blot and immunostaining provide semi-quantitative rather than absolute quantification of modification levels.

• Temporal resolution: Current methods provide snapshots rather than continuous monitoring of modification dynamics.

Researchers should acknowledge these limitations when designing experiments and interpreting results, incorporating additional complementary approaches when possible.

What future technological developments might enhance β-hydroxybutyrylation research?

Several emerging technologies and approaches hold promise for advancing β-hydroxybutyrylation research:

• Advanced antibody engineering:

  • Development of recombinant antibodies with enhanced specificity

  • Creation of nanobodies or aptamers with improved epitope access

  • Site-specific antibodies for different histone variants

• Live-cell epigenetic imaging:

  • CRISPR-based visualization of modified histones in real-time

  • Development of modification-specific fluorescent probes

  • Super-resolution microscopy techniques for subnuclear localization

• Single-cell epigenomics:

  • Adaptation of CUT&Tag or CUT&RUN for single-cell analysis of β-hydroxybutyrylation

  • Integration with single-cell transcriptomics

  • Spatial epigenomics to map modifications in tissue contexts

• Synthetic biology approaches:

  • Designer histones with site-specific modifications

  • Orthogonal enzymatic systems for targeted modification

  • Optogenetic control of β-hydroxybutyrylation enzymes

• Computational advances:

  • Machine learning algorithms to predict β-hydroxybutyrylation sites

  • Network analysis tools to integrate modification data with metabolomics

  • Structural modeling of modification effects on chromatin architecture

• Metabolic engineering:

  • Cell-type specific manipulation of ketone metabolism

  • Targeted delivery of β-hydroxybutyrate to specific tissues

  • Development of β-hydroxybutyrylation-specific metabolic inhibitors