2-hydroxyisobutyryl-HIST1H4A (K91) Antibody

Basic Research Questions

• What is 2-hydroxyisobutyrylation and why is it significant in histone biology?

  2-hydroxyisobutyrylation is a recently identified post-translational modification that occurs on lysine residues of proteins, including histones. This modification affects the association between histone and DNA, playing a crucial role in regulating chromatin structure and gene expression . Research published in Nature has demonstrated that this modification is highly conserved across species and is broadly obtainable in plant cells, suggesting its fundamental role in cellular processes . Beyond histones, studies have identified thousands of 2-hydroxyisobutyryl lysine sites on non-histone proteins (9,916 sites on 2,512 proteins in developing rice seeds), indicating this modification has widespread regulatory functions across the proteome .

• How does 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody differ from antibodies targeting other lysine positions?

  The 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody specifically recognizes the 2-hydroxyisobutyrylation modification at lysine 91 (K91) of the Histone H4 protein. According to product information, this antibody is generated using a synthesized peptide derived from Human Histone H4 protein specifically encompassing amino acids 85-96, which contains the K91 residue . This specificity distinguishes it from antibodies targeting other modified lysine positions such as K12, K31, or K77 on Histone H4, which recognize the same type of modification but at different functional positions.

  Antibody Target	Immunogen	Applications	Host Species	Reference
  K91	Peptide from Human Histone H4 (85-96aa)	ELISA, ICC	Rabbit
  K77	Peptide around site of K77	ELISA, WB	Rabbit
  K31	Peptide around site of K31	ELISA, WB, IF	Rabbit
  K12	Not specified in results	ChIP, ELISA, ICC, IF, IP, WB	Rabbit

• What are the primary applications of 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody in research?

  Based on the available data, 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody is utilized in multiple experimental approaches to study this specific histone modification:

  Application	Description	Typical Dilution/Concentration	Data Obtained
  ELISA	Quantitative detection of the modification in protein samples	Optimal dilutions determined by end user	Quantitative measurement of modification levels
  ICC (Immunocytochemistry)	Visualization of cellular localization of the modification	Not specified for K91	Spatial distribution of the modification within cells
  Western Blot (WB)	Detecting the modified protein in cell lysates	1:100-1:1000 (based on similar antibodies)	Verification of modification and relative abundance
  ChIP	Analysis of genomic distribution of the modification	Not specified for K91	Genomic locations enriched for the modification

  Researchers should optimize these applications for their specific experimental systems through preliminary titration experiments .

• What is the role of Histone H4 in chromatin structure and gene regulation?

  Histone H4 is a core component of nucleosomes, the fundamental units of chromatin. According to the product information, nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template . This structural role makes Histone H4 central to transcription regulation, DNA repair, DNA replication, and chromosomal stability.

  DNA accessibility is regulated through a complex set of post-translational modifications of histones, collectively known as the histone code, along with nucleosome remodeling . The specific modifications on Histone H4, including 2-hydroxyisobutyrylation at K91, contribute to this regulatory code by altering the electrostatic interactions between histones and DNA, and by providing binding sites for effector proteins that influence chromatin structure and function.

• How does 2-hydroxyisobutyrylation compare to other post-translational modifications on histones?

  2-hydroxyisobutyrylation represents one of many post-translational modifications that occur on histone proteins. While the search results provide limited comparative data, we can outline key distinctions:

  Modification	Chemical Group	Common Positions on H4	Known Functions	Detection Methods
  2-hydroxyisobutyrylation	2-hydroxyisobutyryl group	K12, K31, K77, K91	Affects histone-DNA association	Antibody-based methods, mass spectrometry
  Acetylation	Acetyl group	K5, K8, K12, K16	Gene activation, chromatin decondensation	Antibody-based methods, mass spectrometry
  Methylation	Methyl group(s)	K20	Transcriptional regulation, DNA repair	Antibody-based methods, mass spectrometry
  Phosphorylation	Phosphate group	S1	Mitosis, DNA damage response	Antibody-based methods, mass spectrometry

  According to the Nature article, lysine 2-hydroxyisobutyrylation is highly conserved across species, suggesting its fundamental importance in cellular processes . This modification is broadly distributed in plant cells, indicating a widespread regulatory function similar to other well-studied histone modifications .

Advanced Research Questions

• What are the optimal experimental conditions for using 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody in ChIP assays?

  While specific ChIP protocols for the K91 antibody are not provided in the search results, optimal conditions can be inferred from similar antibodies and general ChIP practices:

  Experimental Step	Recommended Conditions	Important Considerations
  Fixation	1% formaldehyde, 10 minutes, RT	Over-fixation can mask epitopes
  Sonication	Optimize to obtain 200-500 bp fragments	Verify fragmentation by gel electrophoresis
  Antibody amount	2-5 μg per ChIP	Titrate for optimal signal-to-noise ratio
  Incubation	Overnight at 4°C with gentle rotation	Longer incubation improves enrichment
  Controls	Include IgG control and input samples	Essential for data normalization
  Validation	Consider sodium butyrate treatment (30mM, 4h)	Increases histone modifications as shown for similar antibodies

  Researchers should validate these conditions for their specific experimental system and biological question, particularly as the antibody's performance may vary across different cell types and chromatin preparations.

• How can researchers validate the specificity of 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody?

  Comprehensive validation of antibody specificity is critical for reliable experimental results. Researchers should implement multiple complementary approaches:

  Validation Method	Experimental Approach	Expected Outcome
  Peptide competition assay	Pre-incubate antibody with modified and unmodified peptides	Signal should be blocked by K91-modified peptide but not by unmodified or other modified peptides
  Western blot analysis	Test multiple cell lines with/without HDAC inhibitor treatment	Should detect specific band at ~12 kDa (as observed with similar H4 antibodies)
  Dot blot peptide array	Test against modified and unmodified peptides	Should only recognize K91-modified peptide
  Immunoprecipitation-mass spectrometry	Analyze proteins pulled down by the antibody	Should enrich for H4 with K91 modification
  CRISPR-Cas9 K91R mutation	Generate lysine-to-arginine mutation at position 91	Should eliminate antibody binding
  Treatment with sodium butyrate	30mM for 4h as shown for other histone antibodies	Should increase modification level

  Methodical validation using these approaches ensures experimental results accurately reflect the biological reality of 2-hydroxyisobutyrylation at K91 of Histone H4.

• What are the technical challenges in detecting 2-hydroxyisobutyrylation across different cell types and species?

  Researchers face several technical challenges when studying 2-hydroxyisobutyrylation in diverse biological systems:

  Cross-reactivity considerations: The 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody is generated against human Histone H4 , requiring validation for use in other species despite the high conservation of this modification . The surrounding sequence context may vary between species, potentially affecting antibody recognition even if the modification itself is conserved.

  Preservation of modifications: The labile nature of some post-translational modifications necessitates careful sample preparation to prevent loss during extraction and processing. Protease inhibitors and deacetylase inhibitors may be needed to maintain modification integrity.

  Quantitative limitations: Relative abundance of the modification may vary substantially between cell types or conditions, requiring sensitive detection methods and appropriate normalization strategies. Establishing reliable quantification methods remains challenging, particularly for low-abundance modifications.

  Background signal management: Non-specific binding of the antibody can generate background signal, especially in immunofluorescence applications, necessitating stringent washing protocols and appropriate negative controls.

• How does the 2-hydroxyisobutyrylation pattern change during different cellular processes or stress conditions?

  While the search results don't provide specific data on dynamic changes of 2-hydroxyisobutyrylation at K91 under different conditions, existing knowledge suggests several probable patterns:

  Cellular Process/Condition	Expected Change	Experimental Approach to Monitor
  Cell cycle progression	Phase-specific regulation	Synchronize cells, collect at different phases
  Differentiation	Cell type-specific patterns	Compare stem cells to differentiated cells
  Metabolic stress	Altered modification levels	Glucose deprivation, hypoxia treatments
  HDAC inhibition	Increased modification	Sodium butyrate treatment (30mM, 4h)
  DNA damage response	Potential regulatory role	UV irradiation, genotoxic agent exposure

  The observation that sodium butyrate treatment increases histone modifications in cell lysates suggests that inhibiting deacetylases might also affect 2-hydroxyisobutyrylation levels, pointing to potential cross-talk between different histone modifications.

• What are the current limitations in interpreting data from 2-hydroxyisobutyryl-HIST1H4A (K91) Antibody experiments?

  Several limitations constrain the interpretation of 2-hydroxyisobutyrylation data:

  Incomplete understanding of regulatory enzymes: Unlike well-characterized modifications such as acetylation, the enzymes that add ("writers") and remove ("erasers") 2-hydroxyisobutyrylation are not fully identified and characterized, limiting mechanistic interpretations of the data.

  Limited knowledge of biological significance: While the modification affects histone-DNA interactions , the specific functional consequences of 2-hydroxyisobutyrylation at K91 are not comprehensively understood, making it difficult to contextualize experimental findings.

  Potential epitope masking: Adjacent or nearby modifications may influence antibody recognition, potentially leading to false negative results when certain combinations of modifications are present.

  Technical variability: Batch-to-batch variability in antibody production can influence experimental reproducibility, requiring stringent validation for each new lot.

  Researchers should acknowledge these limitations when designing experiments and interpreting results, incorporating appropriate controls to mitigate these challenges.

• How can researchers integrate 2-hydroxyisobutyrylation data with other epigenetic modifications to understand the histone code?

  Integration of multiple histone modification datasets provides comprehensive insights into chromatin regulation:

  Integration Approach	Methodology	Insights Gained
  Sequential ChIP (Re-ChIP)	Perform ChIP with first antibody, then second	Co-occurrence of modifications on same nucleosomes
  Multi-omics correlation	Integrate ChIP-seq, RNA-seq, ATAC-seq data	Relationship between modifications and gene expression
  Proteomics analysis	Mass spectrometry of histones	Combinatorial patterns of modifications
  Machine learning	Apply algorithms to identify patterns	Predictive models of modification functions
  Temporal dynamics	Time-course experiments	Sequential ordering of modifications
  Perturbation analysis	Inhibit specific enzymes	Causal relationships between modifications

  This integrated approach can elucidate how 2-hydroxyisobutyrylation at K91 operates within the broader context of the histone code, potentially revealing synergistic or antagonistic relationships with other modifications.

• What are the recommended controls and normalization methods for quantitative analysis of 2-hydroxyisobutyrylation?

  Robust quantitative analysis of 2-hydroxyisobutyrylation requires comprehensive controls and normalization:

  Experimental Technique	Essential Controls	Recommended Normalization
  Western Blot	IgG negative control, Total H4 for loading	Normalize to total H4 signal
  ChIP-qPCR	IgG ChIP, Input DNA (1-5%)	Percent input method or normalization to housekeeping gene regions
  ChIP-seq	Input DNA, IgG ChIP, Spike-in controls	RPKM/FPKM normalization, Spike-in normalization
  Immunofluorescence	Secondary antibody only, Peptide competition	Total nuclear intensity or total H4 staining
  Mass Spectrometry	Unmodified peptides, Synthetic standards	Label-free quantification or SILAC

  For Western blot specifically, researchers using 2-hydroxyisobutyryl-HIST1H4A antibodies have detected bands at the expected size of 12 kDa in multiple cell lines, including Hela, A549, K562, and HepG2 . Treatment with sodium butyrate (30mM for 4h) can serve as a positive control by increasing histone modification levels , providing a reference point for quantitative comparisons.