2-hydroxyisobutyryl-HIST1H1C (K22) Antibody

What is 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody and what does it detect?

The 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody is a polyclonal antibody developed in rabbits that specifically recognizes the 2-hydroxyisobutyryl post-translational modification at lysine 22 (K22) of the human Histone H1.2 protein (HIST1H1C). This antibody was developed using a synthetic peptide containing the 2-hydroxyisobutyryl-lysine at position 22 as the immunogen .

Lysine 2-hydroxyisobutyrylation is a relatively recently discovered histone post-translational modification that plays significant roles in epigenetic regulation. The antibody allows researchers to specifically detect this modification at the K22 position of HIST1H1C without cross-reactivity to other modifications or the same modification at different lysine residues .

How does lysine 2-hydroxyisobutyrylation differ from other histone modifications?

Lysine 2-hydroxyisobutyrylation (Khib) differs from better-known modifications like acetylation or methylation in several key ways:

• Chemical structure: The 2-hydroxyisobutyryl group is bulkier than acetyl groups and contains a hydroxyl group that affects its chemical properties

• Metabolic connection: 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases, suggesting this modification may connect metabolism with epigenetic regulation

• Function: While acetylation generally correlates with gene activation and methylation can be associated with either activation or repression depending on the site, 2-hydroxyisobutyrylation appears to have distinct functions in chromatin regulation

• Distribution: Khib occurs on specific lysine residues that may or may not overlap with sites of other modifications, creating unique combinatorial patterns

This modification has been observed in both eukaryotic and prokaryotic proteins, suggesting it's an evolutionarily conserved regulatory mechanism .

What are the validated applications for this antibody?

The 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody has been validated for the following applications:

*Similar antibodies targeting different lysine positions typically use dilutions of 1:2000-1:10000 for ELISA applications .

The antibody is supplied as a liquid in a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. For optimal results, it should be stored at -20°C or -80°C, avoiding repeated freeze-thaw cycles .

What is the optimal protocol for immunofluorescence experiments using this antibody?

For optimal immunofluorescence results with the 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody:

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

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

• Permeabilize with 0.2% Triton X-100 in PBS for 5-10 minutes

• Block with 5% normal serum in PBS with 0.1% Tween-20 for 1 hour at room temperature

• Incubate with primary antibody at 1:50-1:200 dilution in blocking buffer overnight at 4°C

• Wash 3x with PBS containing 0.1% Tween-20

• Incubate with fluorophore-conjugated anti-rabbit secondary antibody for 1 hour at room temperature

• Wash 3x with PBS containing 0.1% Tween-20

• Counterstain nuclei with DAPI and mount with anti-fade mounting medium

• Image using confocal or fluorescence microscopy

Critical considerations include using appropriate controls (primary antibody omission, blocking peptide competition) and optimizing antibody concentration for your specific cell type and fixation method .

How should samples be prepared to maximize detection of 2-hydroxyisobutyryl-HIST1H1C (K22)?

To maximize detection of the 2-hydroxyisobutyryl modification at K22 of HIST1H1C:

• Consider cellular treatments that enhance 2-hydroxyisobutyrylation:

  • Treatment with histone deacetylase inhibitors like sodium butyrate (30mM for 4 hours) can increase certain histone modifications

  • Metabolic interventions that alter cellular 2-hydroxyisobutyric acid levels may affect modification levels

• Optimize protein extraction:

  • For histones, use acid extraction methods (e.g., 0.2N HCl) to efficiently isolate histones

  • Include deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) in lysis buffers

  • Add protease inhibitors to prevent degradation

• For fixed samples:

  • Use fresh fixatives and control fixation time carefully

  • Consider epitope retrieval methods if initial detection is weak

  • Optimize permeabilization to ensure antibody access to nuclear proteins

• Timing considerations:

  • This modification may be dynamic, so standardize the timing of sample collection

  • Consider performing time-course experiments to capture modification dynamics

These approaches should increase the likelihood of detecting the target modification while maintaining its natural state .

What controls are essential when using the 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody?

Essential controls for experiments using this antibody include:

Incorporating these controls is essential for reliable data interpretation and validation of experimental findings .

How should Western blot data using this antibody be quantified and normalized?

For accurate quantification and normalization of Western blot data:

• Signal quantification:

  • Use digital imaging systems with a linear dynamic range

  • Capture images before signal saturation occurs

  • Apply appropriate background subtraction methods

  • Measure integrated density of bands rather than just peak intensity

• Normalization strategies:

  • Primary normalization to total HIST1H1C protein levels (using a total HIST1H1C antibody)

  • Secondary normalization to other histone proteins (H3 or H4) for loading control

  • Consider normalizing to the sum of all histone H1 variants detected

• Experimental comparisons:

  • Always include a reference sample on each blot for inter-blot comparisons

  • For time-course experiments, express data as fold-change from baseline

  • For treatment comparisons, normalize to vehicle control samples

• Statistical analysis:

  • Perform quantification from at least three biological replicates

  • Apply appropriate statistical tests (t-test for paired comparisons, ANOVA for multiple comparisons)

  • Present data with error bars representing standard deviation or standard error

This methodical approach ensures reliable quantitative analysis of the 2-hydroxyisobutyryl modification at K22 of HIST1H1C .

What are common technical issues with this antibody and their solutions?

Troubleshooting should systematically address each possible cause while maintaining appropriate controls to validate the results .

How can ChIP-seq data using this antibody be analyzed and interpreted?

While the search results don't specifically mention ChIP validation for the K22 antibody, similar antibodies (like K116) are validated for ChIP . For ChIP-seq data analysis and interpretation:

• Quality control and preprocessing:

  • Assess sequencing quality metrics (base quality scores, GC content)

  • Filter out low-quality reads and remove adapter sequences

  • Align to reference genome using appropriate alignment parameters

• Peak calling and annotation:

  • Use peak calling algorithms suitable for histone modifications (e.g., MACS2)

  • Identify genomic regions enriched for K22 2-hydroxyisobutyrylation

  • Annotate peaks to genomic features (promoters, enhancers, gene bodies)

• Comparative analysis:

  • Compare K22 2-hydroxyisobutyrylation patterns with other histone modifications

  • Correlate with gene expression data from RNA-seq

  • Analyze differences between experimental conditions

• Functional interpretation:

  • Perform gene ontology and pathway enrichment analysis of modified regions

  • Identify transcription factor binding motifs within or near peaks

  • Integrate with chromatin accessibility data (ATAC-seq, DNase-seq)

• Visualization:

  • Generate genome browser tracks for interactive exploration

  • Create heatmaps and profile plots around genomic features

  • Develop circos plots for genome-wide visualization

This comprehensive analysis approach can reveal the genomic distribution and potential functional implications of K22 2-hydroxyisobutyrylation .

How does 2-hydroxyisobutyrylation at K22 potentially relate to metabolic diseases?

The connection between 2-hydroxyisobutyrylation at K22 of HIST1H1C and metabolic diseases is an emerging area of research, with several potential mechanisms:

• Metabolic signaling integration:

  • 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases

  • K22 2-hydroxyisobutyrylation may serve as a chromatin-level sensor of cellular metabolic state

  • This modification could translate metabolic signals into altered gene expression patterns

• Regulatory mechanisms:

  • Enzymes responsible for adding or removing this modification might be metabolically regulated

  • Cellular concentrations of 2-HIBA or its precursors may influence modification levels

  • Metabolic pathways involving 2-HIBA could affect chromatin structure via this modification

• Disease implications:

  • Altered K22 2-hydroxyisobutyrylation patterns might contribute to metabolic disease pathogenesis

  • This modification could regulate genes involved in glucose metabolism, lipid homeostasis, or insulin signaling

  • Targeting the enzymes involved in this modification might represent therapeutic opportunities

• Comparative studies:

  • Comparing K22 2-hydroxyisobutyrylation levels between healthy and diseased tissues might reveal disease associations

  • Analysis across different metabolic conditions could identify regulatory patterns

Although direct evidence linking K22 2-hydroxyisobutyrylation to specific metabolic diseases is still emerging, the established connection between 2-HIBA and metabolic disorders suggests important functional relationships .

How can multi-omics approaches integrate with 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody studies?

Integration of multi-omics approaches with K22 antibody studies can provide comprehensive insights:

• Genomic integration:

  • ChIP-seq with the K22 antibody maps genome-wide distribution

  • Integration with RNA-seq correlates modification with gene expression

  • ATAC-seq associates the modification with chromatin accessibility states

  • DNA methylation analysis reveals interplay between histone modifications and DNA methylation

• Proteomic connections:

  • Mass spectrometry validates antibody specificity and quantifies modification stoichiometry

  • Proteome-wide analysis identifies other proteins with 2-hydroxyisobutyrylation

  • Interactome studies discover proteins that bind specifically to 2-hydroxyisobutyrylated K22

• Metabolomic relationships:

  • Measure cellular 2-hydroxyisobutyric acid levels and correlate with modification abundance

  • Identify metabolic pathways that influence K22 2-hydroxyisobutyrylation

  • Study the enzymes involved in 2-HIBA metabolism and their relationship to chromatin regulation

• Single-cell approaches:

  • CUT&Tag with the K22 antibody enables single-cell profiling of this modification

  • Integration with scRNA-seq reveals cell-type specific correlations

  • Spatial transcriptomics maps tissue-specific patterns

• Computational integration:

  • Network analysis reveals regulatory relationships

  • Machine learning identifies patterns across datasets

  • Predictive modeling of modification dynamics under different conditions

These integrated approaches provide systems-level understanding of K22 2-hydroxyisobutyrylation in cellular regulation .

What is known about the enzymatic regulation of 2-hydroxyisobutyrylation at K22?

The enzymatic regulation of 2-hydroxyisobutyrylation at K22 of HIST1H1C is not fully characterized, but several mechanisms can be inferred from related research:

• "Writer" enzymes (adding the modification):

  • While specific enzymes for K22 haven't been definitively identified, p300/CBP acetyltransferases have been implicated in catalyzing some 2-hydroxyisobutyrylation reactions

  • Acyl-CoA synthetases like the 2-HIBA-CoA ligase may generate the 2-hydroxyisobutyryl-CoA substrate needed for the modification

  • The reaction likely requires 2-hydroxyisobutyryl-CoA as a donor substrate

• "Eraser" enzymes (removing the modification):

  • Histone deacetylases (HDACs) may remove 2-hydroxyisobutyryl groups

  • Sirtuin family deacylases, particularly SIRT3, have been implicated in removing similar acyl modifications

  • Inhibition of these enzymes with sodium butyrate may explain the increased modification levels after treatment

• "Reader" proteins (recognizing the modification):

  • Bromodomain-containing proteins may recognize 2-hydroxyisobutyrylated lysines

  • YEATS domain proteins have been shown to bind to some acylated lysine residues

  • The specific readers for K22 2-hydroxyisobutyrylation remain to be identified

• Regulation mechanisms:

  • Cellular concentrations of 2-hydroxyisobutyric acid likely influence modification levels

  • Metabolic pathways generating or consuming 2-HIBA may indirectly regulate this modification

  • Competition with other lysine modifications at K22 may provide an additional regulatory layer

Further research using the 2-hydroxyisobutyryl-HIST1H1C (K22) antibody combined with enzymatic studies will help elucidate these regulatory mechanisms .

How do researchers compare 2-hydroxyisobutyryl modifications across different lysine residues in HIST1H1C?

Comparing 2-hydroxyisobutyryl modifications across different lysine residues (such as K22, K116, and K128) in HIST1H1C requires sophisticated technical approaches:

• Antibody-based comparative analysis:

  • Use site-specific antibodies for each lysine position (K22, K116, K128)

  • Apply identical experimental conditions and standardized protocols

  • Include calibration standards to account for different antibody affinities

  • Perform parallel experiments with the same samples across all antibodies

• Mass spectrometry approaches:

  • Bottom-up proteomics with enzymatic digestion to generate lysine-containing peptides

  • Middle-down approaches analyzing larger histone fragments

  • Parallel reaction monitoring (PRM) for targeted quantification of specific modified sites

  • Crosslinking mass spectrometry to understand structural implications of modifications at different sites

• Functional comparisons:

  • ChIP-seq with each site-specific antibody to compare genomic distributions

  • Mutational studies replacing specific lysines to prevent modification

  • Correlation of each site's modification with gene expression data

  • Computational modeling of structural impacts of modifications at different residues

• Dynamic analysis:

  • Time-course experiments to compare modification kinetics across sites

  • Response to treatments that alter modification levels

  • Recovery dynamics after inhibitor removal

These approaches allow researchers to determine site-specific functions and regulatory mechanisms of 2-hydroxyisobutyrylation across different lysine residues in HIST1H1C .

What computational approaches enhance research using the 2-hydroxyisobutyryl-HIST1H1C (K22) Antibody?

Advanced computational methods significantly enhance research with this antibody:

• Structural analysis:

  • Molecular dynamics simulations of HIST1H1C with and without K22 2-hydroxyisobutyrylation

  • Prediction of conformational changes induced by the modification

  • Modeling of interactions between modified K22 and potential binding partners

  • Analysis of electrostatic surface changes caused by the modification

• Genome-wide data analysis:

  • Specialized peak calling algorithms for ChIP-seq data

  • Motif discovery to identify DNA sequences associated with the modification

  • Integrative analysis with other histone modifications to identify combinatorial patterns

  • Machine learning to predict target genes based on modification patterns

• Network analysis:

  • Construction of gene regulatory networks centered on K22 2-hydroxyisobutyrylation

  • Pathway enrichment analysis of genes affected by the modification

  • Identification of transcription factors associated with modified regions

  • Cross-talk analysis with other epigenetic modifications

• Evolutionary analysis:

  • Conservation analysis of K22 across species and histone variants

  • Phylogenetic studies of enzymes involved in 2-hydroxyisobutyrylation

  • Comparative genomics of modification patterns across species

• Data integration frameworks:

  • Multi-omics data integration platforms connecting modification data with transcriptomics, proteomics, and metabolomics

  • Visualization tools for complex epigenetic datasets

  • Statistical methods for correlation analysis across different data types

These computational approaches transform antibody-generated data into mechanistic insights about K22 2-hydroxyisobutyrylation's biological functions .