2-hydroxyisobutyryl-HIST1H3A (K36) Antibody

What is 2-hydroxyisobutyrylation of histone H3.1 and what biological role does it play?

2-hydroxyisobutyrylation (2-hib) is a relatively newly discovered post-translational modification of histones that plays a fundamental role in epigenetic regulation. When applied to lysine residues on histone H3.1 (HIST1H3A), particularly at position K36, this modification influences chromatin structure and accessibility, ultimately affecting gene expression patterns. Like other histone modifications, 2-hydroxyisobutyrylation serves as a specific epigenetic mark that helps establish the regulatory landscape of the genome, contributing to DNA packaging, transcriptional regulation, and genomic integrity . Unlike more common modifications such as acetylation or methylation, 2-hydroxyisobutyrylation appears to have distinct regulatory functions and temporal dynamics in cellular processes.

How does 2-hydroxyisobutyryl-HIST1H3A (K36) differ from modifications at other lysine residues?

The 2-hydroxyisobutyryl modification at K36 of histone H3.1 occurs within a region that is functionally distinct from other commonly modified residues. Unlike K18 modifications that typically associate with transcriptional activation , or K56 modifications that relate to nucleosome assembly and DNA damage response , K36 modifications often correlate with transcriptional elongation and intragenic regions of actively transcribed genes. The specific placement of this modification creates a unique microenvironment on the nucleosome surface, recruiting different effector proteins compared to other modified positions. This positional specificity allows for precise regulation of distinct cellular processes.

What are the enzymes responsible for writing and erasing 2-hydroxyisobutyryl marks on HIST1H3A?

The enzymatic machinery responsible for establishing (writing) and removing (erasing) 2-hydroxyisobutyryl marks differs from those handling other histone modifications. While the complete enzyme repertoire is still being characterized, current evidence suggests involvement of specific acetyltransferase family members as "writers" with catalytic promiscuity. The "erasers" likely include members of the histone deacetylase family with broad substrate specificity. The regulatory balance between these enzymes determines the steady-state levels of 2-hydroxyisobutyrylation at K36 and consequently affects downstream biological processes . Understanding these enzymatic pathways provides potential targets for modulating epigenetic states in research or therapeutic contexts.

What are the optimal conditions for using 2-hydroxyisobutyryl-HIST1H3A (K36) antibody in Western blot applications?

For optimal Western blot results using the 2-hydroxyisobutyryl-HIST1H3A (K36) antibody, researchers should:

• Use freshly prepared histone extracts or nuclear fractions to preserve modification integrity

• Apply recommended dilutions (typically 1:500-1:1000 for Western blot based on similar antibodies)

• Block with 5% BSA rather than milk (which contains proteins that may interfere with histone antibody binding)

• Include modification-specific controls alongside unmodified histone controls

• Optimize transfer conditions for histones (which are small, basic proteins)

• Consider using PVDF membranes for better protein retention

• Include phosphatase and deacetylase inhibitors in extraction buffers to prevent modification loss

The antibody's specificity should be verified using peptide competition assays to confirm binding to the modified K36 residue.

How can I optimize immunofluorescence protocols for detecting 2-hydroxyisobutyryl-HIST1H3A (K36) in tissue samples?

Optimizing immunofluorescence for 2-hydroxyisobutyryl-HIST1H3A (K36) detection requires:

• Immediate fixation of samples to preserve modification integrity (delayed fixation can result in significant loss of signal)

• Moderate fixation times (excessive fixation can mask epitopes)

• Appropriate dilution range (1:10-1:200 based on similar antibodies)

• Antigen retrieval optimization (typically citrate buffer pH 6.0)

• Permeabilization with 0.2-0.5% Triton X-100

• Blocking with normal serum matching the secondary antibody host

• Incubation at 4°C overnight for maximal signal specificity

• Inclusion of appropriate controls (both positive and negative)

The heterogeneous staining pattern observed in some histone modification antibodies may reflect cell cycle-specific regulation or differential chromatin states .

What are the critical considerations for ChIP experiments using 2-hydroxyisobutyryl-HIST1H3A (K36) antibody?

Successful ChIP experiments with 2-hydroxyisobutyryl-HIST1H3A (K36) antibody require:

Combining ChIP with high-throughput sequencing (ChIP-seq) allows genome-wide profiling of the 2-hydroxyisobutyryl-K36 distribution across regulatory regions.

How can I verify the specificity of 2-hydroxyisobutyryl-HIST1H3A (K36) antibody against other histone modifications?

Verifying antibody specificity requires multiple approaches:

• Peptide competition assays with modified and unmodified peptides spanning the K36 region

• Dot blot analysis with peptide arrays containing various histone modifications

• Western blot comparison using recombinant histones with defined modifications

• Testing reactivity in cell lines with genetically modified K36 residues (K36R mutants)

• Mass spectrometry validation of immunoprecipitated proteins

• Cross-reactivity assessment with closely related modifications (acetylation, β-hydroxybutyrylation)

Notably, antibodies recognizing 2-hydroxyisobutyryl modifications should be tested against acetylated residues due to structural similarities between these modifications.

What cell types or tissues show significant 2-hydroxyisobutyryl-HIST1H3A (K36) enrichment?

Based on studies of related histone modifications:

• Metabolically active tissues (liver, kidney, heart) typically show higher levels of acylation modifications including 2-hydroxyisobutyrylation

• Developing embryonic tissues during periods of dynamic transcriptional regulation

• Proliferating cells, particularly during S-phase when histones are newly synthesized

• Specialized cell types undergoing metabolic reprogramming (activated immune cells, differentiating stem cells)

• Cancer cells with altered metabolic profiles

The tissue-specific distribution of 2-hydroxyisobutyrylation reflects the availability of metabolic substrates and enzymatic machinery in different cellular contexts. Researchers should consider this when selecting experimental models.

How does cell cycle progression affect 2-hydroxyisobutyryl-HIST1H3A (K36) patterns?

The dynamics of 2-hydroxyisobutyryl-HIST1H3A (K36) across the cell cycle follows patterns similar to other histone modifications but with distinctive features:

• During DNA replication (S-phase), newly synthesized histones undergo establishment of modification patterns

• Certain histone modifications show cell cycle-specific distribution with enrichment in specific phases

• Mitotic cells often display altered histone modification patterns due to chromatin condensation

• The heterogeneous staining observed in cell populations may reflect different cell cycle stages

• The modification may be temporarily reduced during mitosis when chromatin is highly condensed

When designing experiments to detect 2-hydroxyisobutyryl-HIST1H3A (K36), researchers should consider synchronizing cells or using cell cycle markers for appropriate interpretation of results.

How can 2-hydroxyisobutyryl-HIST1H3A (K36) antibody be used to study metabolic-epigenetic connections?

The 2-hydroxyisobutyryl modification represents a direct link between cellular metabolism and epigenetic regulation:

• Researchers can use the antibody to track changes in histone modifications following metabolic perturbations

• Combined with metabolic profiling, the antibody enables correlation between substrate availability and modification levels

• ChIP-seq with the antibody allows mapping of genomic regions sensitive to metabolic states

• In disease models characterized by metabolic dysregulation, the antibody can identify epigenetic consequences

• Time-course experiments during metabolic transitions can reveal dynamic regulation of this modification

This application area represents a frontier in understanding how cellular metabolic state influences gene expression through epigenetic mechanisms.

What are the technical challenges in multiplexing 2-hydroxyisobutyryl-HIST1H3A (K36) detection with other histone modifications?

Multiplexed detection presents several challenges:

• Antibody cross-reactivity between similar modifications must be rigorously controlled

• Species compatibility of primary antibodies must be considered (using antibodies raised in different host species)

• Signal separation requires careful optimization of fluorophores or chromogens with distinct spectral properties

• Sequential detection protocols may be necessary to prevent interference

• Epitope masking can occur when multiple antibodies target nearby residues

• Quantitative assessment becomes more complex due to potential signal interference

Despite these challenges, multiplexed detection offers valuable insights into the combinatorial patterns of histone modifications that constitute the "histone code."

How does 2-hydroxyisobutyryl-HIST1H3A (K36) compare with β-hydroxybutyryl-HIST1H3A (K18) in functional genomics studies?

These related but distinct modifications show important differences:

• Both modifications are metabolically sensitive but likely respond to different metabolic pathways and substrates

• The 2-hydroxyisobutyryl-K36 and β-hydroxybutyryl-K18 modifications occupy different regions of the histone tail, potentially affecting distinct protein-protein interactions

• Genomic distribution analysis reveals different patterns of enrichment across regulatory elements

• Temporal dynamics during cellular processes may differ significantly

• Reader proteins recognizing these modifications likely belong to different effector families

• Functional outcomes in terms of gene expression regulation may be distinct

Comparative studies using both antibodies can provide insights into how cells utilize different metabolite-derived modifications to fine-tune chromatin regulation.

What might cause inconsistent staining patterns when using 2-hydroxyisobutyryl-HIST1H3A (K36) antibody in immunofluorescence?

Inconsistent staining patterns may result from:

• Heterogeneous cell populations at different cell cycle stages or metabolic states

• Variable fixation times affecting epitope preservation (significant signal reduction can occur with delayed or prolonged fixation)

• Inadequate permeabilization preventing antibody access to nuclear epitopes

• Buffer pH variations affecting epitope conformation and antibody binding

• Enzymatic activities in samples removing modifications prior to complete fixation

• Variations in the expression or activity of writers/erasers across cells

• Technical factors such as uneven antibody distribution during incubation

To address these issues, standardize sample handling protocols, particularly fixation timing and conditions, and consider including cellular markers to identify subpopulations.

How can I validate antibody lot consistency for 2-hydroxyisobutyryl-HIST1H3A (K36) detection?

Antibody lot validation should include:

• Side-by-side testing of new and reference lots using the same samples and protocols

• Peptide competition assays to confirm specificity for the modified epitope

• Western blot analysis to verify band pattern and intensity

• Immunofluorescence spatial pattern comparison in control samples

• ChIP-qPCR at known target regions to confirm enrichment consistency

• Record-keeping of lot-specific optimal dilutions and conditions

• Documentation of batch-specific background levels and signal-to-noise ratios

Maintaining reference samples is crucial for comparative analysis across antibody lots, ensuring experimental reproducibility over time.

What factors might cause loss of 2-hydroxyisobutyryl-HIST1H3A (K36) signal in fixed tissues?

Signal loss in fixed tissues may occur due to:

• Delayed fixation leading to enzymatic removal of modifications (>30% reduction can occur after just 2 hours of delay)

• Overfixation masking epitopes or altering their conformation

• Improper storage of fixed tissues

• Dephosphorylation or deacylation by endogenous enzymes prior to complete fixation

• Harsh antigen retrieval conditions degrading the modification

• Inappropriate pH during processing affecting epitope stability

• Multiple freeze-thaw cycles of tissue sections

To preserve modification integrity, immediate fixation with standardized protocols is essential, along with the inclusion of appropriate enzyme inhibitors during sample preparation.