2-hydroxyisobutyryl-HIST1H4A (K5) Antibody

Basic Research Questions

• What is 2-hydroxyisobutyryl-HIST1H4A (K5) antibody and what does it detect?

  The 2-hydroxyisobutyryl-HIST1H4A (K5) antibody is a rabbit polyclonal antibody specifically designed to detect the 2-hydroxyisobutyrylation modification at lysine 5 of histone H4 in human samples. This antibody recognizes a peptide sequence around the site of 2-hydroxyisobutyryl-Lys (5) derived from Human Histone H4. It is purified by antigen affinity chromatography and is typically supplied in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol . The antibody enables researchers to study this specific post-translational modification (PTM) which has been implicated in transcriptional regulation and other epigenetic processes.

• What applications is the 2-hydroxyisobutyryl-HIST1H4A (K5) antibody validated for?

  The 2-hydroxyisobutyryl-HIST1H4A (K5) antibody has been validated for multiple laboratory applications including:

  • ELISA (Enzyme-Linked Immunosorbent Assay)

  • Western Blotting (WB)

  • Immunofluorescence (IF)

  • Immunocytochemistry (ICC)

  When designing experiments, it's important to empirically determine optimal dilutions for each application as these may vary depending on sample type, detection method, and experimental conditions. For Western blotting applications, researchers should expect to detect a band at approximately 11 kDa, which corresponds to the molecular weight of histone H4 .

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

  2-hydroxyisobutyrylation (Khib) is structurally and mechanistically distinct from other histone acylations such as acetylation. While lysine acetylation and 2-hydroxyisobutyrylation both occur on lysine residues, they have:

  • Different chemical structures: 2-hydroxyisobutyrylation has a larger and more complex modification group

  • Distinct genomic distributions: Studies show Khib has unique patterns compared to acetylation marks

  • Different regulatory enzymes: While some histone deacetylases (HDAC 1-3, Rpd3p, and Hos3p) may function as de-2-hydroxyisobutyrylation enzymes, the regulation differs

  • Unique functional impacts: Khib can affect protein-protein interactions differently than acetylation

  Research has shown that histone H4K5 2-hydroxyisobutyrylation, unlike acetylation, can abolish the interaction between the first bromodomain of Brdt and the histone H4 tail, indicating distinct functional consequences .

• What is the biological significance of histone H4K5 2-hydroxyisobutyrylation?

  Histone H4K5 2-hydroxyisobutyrylation represents an important epigenetic modification with several biological functions:

  • Transcriptional regulation: It is associated with active gene transcription

  • Chromatin structure modulation: It affects the interaction between histones and DNA

  • Metabolic sensing: Research indicates it is regulated by glucose availability and cellular metabolism

  • Cell differentiation: It shows dynamic changes during male germ cell differentiation

  Studies have demonstrated that 2-hydroxyisobutyrylation of H4K5 can specifically prevent binding of bromodomain-containing proteins like Brdt, suggesting a role in controlling protein recruitment to chromatin. This contrasts with H4K8 2-hydroxyisobutyrylation, which does not abolish such interactions , highlighting the position-specific effects of this modification.

Advanced Research Questions

• How does the 2-hydroxyisobutyryl-HIST1H4A (K5) antibody specificity compare to antibodies for other H4 modifications?

  When evaluating antibody specificity for histone PTMs, cross-reactivity is a critical concern. The 2-hydroxyisobutyryl-HIST1H4A (K5) antibody demonstrates high specificity for its target modification compared to other H4 modification antibodies. Research has shown:

  Antibody	Target Modification	Cross-reactivity with other PTMs	Applications	Reference
  Anti-H4K5hib	2-hydroxyisobutyrylation at K5	Minimal cross-reactivity with H4K5ac	WB, ELISA, IF/ICC
  Anti-H4K5ac	Acetylation at K5	Some cross-reactivity when K8 is acetylated	WB, ChIP-seq
  Anti-H4K5bhb	β-hydroxybutyrylation at K5	Minimal cross-reactivity with other K5 modifications	WB
  Anti-H4K8hib	2-hydroxyisobutyrylation at K8	Minimal cross-reactivity with H4K8ac	WB, ICC/IF

  The CMA405 antibody for H4K5ac shows a unique property of only reacting with K5ac when the neighboring K8 is unacetylated, allowing researchers to distinguish between newly assembled H4 (diacetylated at K5 and K12) and hyperacetylated H4 (where both K5 and K8 are acetylated) . Similar detailed characterization for the 2-hydroxyisobutyryl-HIST1H4A (K5) antibody is essential for accurate interpretation of experimental results.

• What are the optimal protocols for using 2-hydroxyisobutyryl-HIST1H4A (K5) antibody in ChIP-seq experiments?

  For successful ChIP-seq experiments using 2-hydroxyisobutyryl-HIST1H4A (K5) antibody, consider the following methodological approach:

  1. Sample preparation:

     • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

     • Quench with 0.125 M glycine for 5 minutes

     • Harvest cells and wash with cold PBS containing protease inhibitors

  2. Chromatin preparation:

     • Lyse cells in appropriate buffers containing protease inhibitors

     • Sonicate chromatin to fragments of 200-500 bp (optimize sonication time)

     • Confirm fragment size by agarose gel electrophoresis

  3. Immunoprecipitation:

     • Pre-clear chromatin with protein A/G beads

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

     • Add protein A/G beads and incubate for 2-3 hours

     • Wash extensively with increasingly stringent buffers

  4. DNA recovery and library preparation:

     • Reverse cross-links at 65°C overnight

     • Purify DNA using standard methods

     • Prepare sequencing library according to platform requirements

  5. Controls and validation:

     • Include input control (non-immunoprecipitated chromatin)

     • Include IgG control for non-specific binding

     • Validate enrichment by qPCR before sequencing

     • Consider using spike-in controls for quantitative comparisons

  Based on studies with similar histone modification antibodies, surface plasmon resonance (SPR) measurements are recommended to determine antibody affinity for the target epitope, and ChIP-qPCR should be performed to confirm significant enrichment at selected genomic regions before proceeding to genome-wide sequencing .

• How do cellular metabolic states affect histone H4K5 2-hydroxyisobutyrylation levels?

  Cellular metabolism has profound effects on histone 2-hydroxyisobutyrylation levels, particularly at H4K5. Research has established several key relationships:

  • Glucose availability: Studies in yeast have shown that histone 2-hydroxyisobutyrylation levels are regulated by glucose concentration, with the modification responding to changes in glycolytic flux

  • Metabolic intermediates: The cellular levels of 2-hydroxyisobutyrate and 2-hydroxyisobutyryl-CoA, which serve as metabolic precursors for this modification, directly impact the extent of histone 2-hydroxyisobutyrylation

  • Nutrient stress response: In plants, histone Khib works together with H3K23ac to regulate genes involved in starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis, helping to fine-tune plant response to dark-induced starvation

  • Enzyme regulation: Both writers (acetyltransferases like Esa1p and TIP60) and erasers (deacetylases like HDAC1-3, Rpd3p, and Hos3p) of this modification are influenced by metabolic states

  Experimental data from yeast studies demonstrated that proteins involved in glycolysis and electron transport chain complexes are differentially 2-hydroxyisobutyrylated under varying glucose concentrations, suggesting a broader metabolic regulatory network beyond histones .

• What is the cross-talk between histone H4K5 2-hydroxyisobutyrylation and other histone modifications?

  The interplay between histone H4K5 2-hydroxyisobutyrylation and other histone modifications reveals complex regulatory mechanisms:

  1. Spatial relationships:

     • In Arabidopsis, histone Khib does not overlap with frequently modified N-tail lysines such as H3K4, H3K9, and H4K8

     • H4K5 2-hydroxyisobutyrylation and acetylation can be mutually exclusive at the same residue

  2. Functional interactions:

     • H4K5 2-hydroxyisobutyrylation abolishes the binding of Brdt to histone H4, while H4K8 2-hydroxyisobutyrylation only slightly affects this interaction

     • In plants, co-enrichment of histone Khib and H3K23ac correlates with high gene expression levels

  3. Enzymatic regulation:

     • Some histone deacetylases (HDACs) can remove both acetyl and 2-hydroxyisobutyryl groups

     • In plants, HDA6 and HDA9 are involved in removing histone Khib

  4. Competitive modifications:

     • Studies on H4K5 indicate complex interactions between different types of acylations (acetylation, propionylation, butyrylation, crotonylation, and 2-hydroxyisobutyrylation)

     • Research with PRMTs (protein arginine methyltransferases) showed that H4K5 2-hydroxyisobutyrylation decreased arginine methylation by PRMT1, PRMT3, and PRMT8, demonstrating cross-regulation between different types of modifications

  This cross-talk creates a complex "histone code" where combinations of modifications fine-tune the cellular response to various stimuli.

• How can I troubleshoot non-specific binding or weak signals when using the 2-hydroxyisobutyryl-HIST1H4A (K5) antibody?

  When encountering issues with 2-hydroxyisobutyryl-HIST1H4A (K5) antibody performance, consider these methodological troubleshooting approaches:

  For weak signals:

  1. Optimization of antibody concentration:

     • Perform a titration experiment using 1:250, 1:1000, and 1:4000 dilutions to identify optimal concentration

     • For ICC/IF applications, starting dilutions of 1:7.5 to 1:15 have been effective for similar histone modification antibodies

  2. Enhancement of target accessibility:

     • Increase cell permeabilization time (up to 15 minutes with 0.3% Triton X-100)

     • For Western blots, ensure complete protein denaturation and optimize transfer conditions for histones

  3. Signal amplification:

     • Consider using a biotin-streptavidin system for detection

     • For ICC, an HRP-conjugated SP system may provide enhanced sensitivity

  For non-specific binding:

  1. Blocking optimization:

     • Use 10% normal goat serum for ICC/IF applications

     • For Western blots, 5% BSA in TBST is often more effective than milk for phospho-specific antibodies

  2. Validation controls:

     • Include peptide competition assays to confirm specificity

     • Compare signals in cells treated with HDAC inhibitors (e.g., sodium butyrate) versus untreated cells

     • Include appropriate negative controls (samples without the modification)

  3. Cross-reactivity testing:

     • Test antibody against synthetic peptides bearing similar modifications (acetylation, butyrylation, etc.)

     • Consider multiplex assays like dCypher Luminex to simultaneously evaluate antibody specificity against multiple modified histone forms

  For robust quantitative comparisons, normalize data to total H4 levels and include multiple biological replicates to account for variability in modification levels.

• What emerging techniques are being developed to study histone H4K5 2-hydroxyisobutyrylation dynamics?

  Several cutting-edge techniques are advancing our understanding of histone H4K5 2-hydroxyisobutyrylation dynamics:

  1. Site-specific incorporation technologies:

     • Amber suppression-mediated strategy allows site-specific incorporation of ε-N-2-Hydroxyisobutyryl-lysine into proteins in bacteria and mammalian cells

     • This approach enables precise control over modification placement for functional studies

  2. Multicolor immunofluorescence:

     • Direct labeling of modification-specific antibodies enables simultaneous visualization of multiple histone modifications in single cells

     • This technique allows temporal tracking of modification dynamics during the cell cycle

  3. Mass spectrometry-based proteomics:

     • Antibody-based affinity enrichment combined with nano-HPLC/MS/MS analysis of 2-hydroxyisobutyrylation peptides

     • This approach has identified thousands of 2-hydroxyisobutyryl lysine sites across diverse proteomes

  4. CRISPR-based epigenome editing:

     • Fusion of catalytic domains of putative 2-hydroxyisobutyrylation writers or erasers to catalytically inactive Cas9 (dCas9)

     • This allows targeted modulation of H4K5hib at specific genomic loci

  5. Stable isotope labeling approaches:

     • SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) combined with mass spectrometry to quantitatively track changes in 2-hydroxyisobutyrylation in response to metabolic alterations

     • This approach has revealed that 2-hydroxyisobutyrylation levels are differentially regulated under different glucose concentrations

  6. Surface plasmon resonance (SPR):

     • Used to measure the affinities of antibodies for their respective targets

     • Enables comparison of binding affinities between different histone modification antibodies

  These emerging technologies provide researchers with powerful tools to investigate the dynamic regulation and functional consequences of histone H4K5 2-hydroxyisobutyrylation in diverse biological contexts.