2-hydroxyisobutyryl-HIST1H4A (K77) Antibody

What is 2-hydroxyisobutyryl-HIST1H4A (K77) and what is its significance in epigenetic regulation?

2-hydroxyisobutyrylation represents a post-translational modification of histones that has been characterized more recently compared to acetylation and methylation. The 2-hydroxyisobutyryl modification at lysine 77 (K77) of histone H4 (HIST1H4A) contributes to the histone code that regulates chromatin structure and gene expression. Histone H4 functions as a core component of nucleosomes, which wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machinery that requires DNA as a template .

Post-translational modifications like 2-hydroxyisobutyrylation alter the physical and chemical properties of histones, affecting DNA-histone interactions and recruiting specific protein complexes that influence transcription, DNA repair, and replication. The 2-hydroxyisobutyryl modification is chemically distinct from acetylation and methylation, suggesting unique functional consequences for chromatin dynamics and gene regulation .

What validated applications is the 2-hydroxyisobutyryl-HIST1H4A (K77) antibody suitable for?

Based on validation studies, the 2-hydroxyisobutyryl-HIST1H4A (K77) antibody has been confirmed for these specific applications:

This polyclonal antibody, raised in rabbit, specifically targets the peptide sequence surrounding the 2-hydroxyisobutyryl-Lys (77) site derived from Human Histone H4 . For applications not explicitly validated, researchers should conduct preliminary experiments to determine optimal conditions and include appropriate controls to confirm specificity.

What are the optimal storage conditions for maintaining 2-hydroxyisobutyryl-HIST1H4A (K77) antibody activity?

To preserve optimal activity and extend shelf life, the 2-hydroxyisobutyryl-HIST1H4A (K77) antibody should be:

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

• Aliquoted upon receipt to avoid repeated freeze/thaw cycles that can degrade antibody quality

• Maintained in the buffer provided (typically 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol)

• Thawed completely before use and mixed gently

For quality assurance purposes, researchers should document lot numbers and receipt dates, particularly when conducting critical experiments requiring high specificity and sensitivity.

What are the critical considerations for Western blot experiments using 2-hydroxyisobutyryl-HIST1H4A (K77) antibody?

Designing effective Western blot experiments with 2-hydroxyisobutyryl-HIST1H4A (K77) antibody requires attention to several critical factors:

Sample preparation:

• Include appropriate extraction buffers with histone deacetylase inhibitors (like sodium butyrate) and protease inhibitors to preserve the 2-hydroxyisobutyryl modification

• Consider acid extraction methods specifically designed for histones to increase target protein yield

• Include positive controls from cells treated with histone modification-preserving agents

Gel electrophoresis and transfer:

• Use high percentage (15-18%) gels optimized for separation of low molecular weight proteins like histones

• Consider specialized transfer conditions for basic proteins

• Use PVDF membranes rather than nitrocellulose for better retention of histone proteins

Antibody incubation:

• Begin with the recommended dilution range (1:500 - 1:2000) and optimize as needed

• Use 5% BSA or milk in TBS-T for blocking (determine empirically which works best)

• Consider overnight incubation at 4°C for optimal results

Detection and quantification:

• Include a loading control like total H4 antibody on the same or parallel blot

• For quantitative analysis, normalize to loading controls

How can I optimize Chromatin Immunoprecipitation (ChIP) protocols for 2-hydroxyisobutyryl-HIST1H4A (K77) antibody?

While specific ChIP validation for 2-hydroxyisobutyryl-HIST1H4A (K77) antibody isn't explicitly mentioned in the provided information, researchers can adapt standard ChIP protocols with these considerations:

Crosslinking and chromatin preparation:

• Use 1% formaldehyde for 10 minutes at room temperature for standard crosslinking

• Consider dual crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde for improved histone PTM capture

• Include histone deacetylase inhibitors (5-10 mM sodium butyrate) in all buffers to preserve 2-hydroxyisobutyryl modifications

• Sonicate chromatin to 200-500 bp fragments for optimal results

Immunoprecipitation:

• Determine optimal antibody concentration through titration experiments (typically 2-5 μg per reaction)

• Include IgG control, input control, and a positive control (antibody against a well-characterized histone mark)

• Extend incubation time to overnight at 4°C with gentle rotation

Analysis methods:

• Quantify enrichment using qPCR for specific targets or genomic regions

• For genome-wide analysis, consider ChIP-seq approaches with appropriate sequencing depth

• Use bioinformatics tools specifically designed for histone modification analysis

What are the methodological challenges in distinguishing 2-hydroxyisobutyrylation from other acylation modifications?

Distinguishing 2-hydroxyisobutyrylation from other acylation modifications presents several technical challenges:

Antibody specificity:
Antibodies must be rigorously validated to ensure they recognize 2-hydroxyisobutyrylation specifically without cross-reactivity to similar modifications like acetylation, propionylation, or butyrylation. This is particularly important since multiple acylation types can occur at the same lysine residue.

Mass spectrometry challenges:
While mass spectrometry can distinguish different acylations based on mass differences, challenges include:

• Similar fragmentation patterns between some acylations

• Low abundance of specific modifications

• The need for high-resolution instruments

• Complex data analysis requirements

Technical approaches for differentiation:

• Chemical derivatization strategies to enhance separation of modifications

• Enrichment methods specific to each modification type

• Multiple reaction monitoring (MRM) mass spectrometry for targeted analysis

• Specialized chromatographic separation before analysis

How can I design experiments to study dynamic changes of H4K77 2-hydroxyisobutyrylation during cell cycle progression?

Studying dynamic changes in H4K77 2-hydroxyisobutyrylation during the cell cycle requires careful experimental design:

Cell synchronization methods:

• Double thymidine block for G1/S boundary synchronization

• Nocodazole treatment for M-phase arrest

• Serum starvation-release for G0/G1 transition

Verification of synchronization:

• Flow cytometry with propidium iodide staining for DNA content

• Western blot for phase-specific markers

• Immunofluorescence for mitotic indices

Analysis methods:

• Western blot with 2-hydroxyisobutyryl-HIST1H4A (K77) antibody for bulk changes

• ChIP-seq at different cell cycle stages to map genomic distribution changes

• Immunofluorescence microscopy to visualize nuclear localization patterns

Since histone H4 modifications are known to undergo drastic changes during the cell cycle , this experimental approach can reveal how H4K77 2-hydroxyisobutyrylation specifically changes throughout different cell cycle phases.

How can I address non-specific binding issues when using 2-hydroxyisobutyryl-HIST1H4A (K77) antibody?

Non-specific binding is a common challenge when working with histone modification antibodies. For the 2-hydroxyisobutyryl-HIST1H4A (K77) antibody, consider these troubleshooting approaches:

Optimize blocking conditions:

• Test different blocking agents (BSA vs. milk)

• Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

• Include 0.1-0.3% Tween-20 in blocking and antibody dilution buffers

Adjust antibody conditions:

• Perform an antibody dilution series to identify optimal concentration

• Reduce primary antibody incubation time or temperature

• Include competing peptides (unmodified) to reduce non-specific binding

Improve washing steps:

• Increase number and duration of washes

• Use higher concentration of Tween-20 (0.1-0.5%) in wash buffers

• Consider more stringent washing buffers for persistent background

Controls to include:

• Peptide competition assay with 2-hydroxyisobutyrylated and non-modified peptides

• Samples known to lack the modification (e.g., specific knockout or inhibitor-treated cells)

What are the key considerations for validating antibody specificity for 2-hydroxyisobutyryl-HIST1H4A (K77)?

Rigorous validation is crucial for ensuring the specificity of histone modification antibodies. For 2-hydroxyisobutyryl-HIST1H4A (K77) antibody, consider these validation methods:

Peptide competition assays:

• Pre-incubate the antibody with increasing concentrations of:

  • 2-hydroxyisobutyrylated H4K77 peptide (should block signal)

  • Unmodified H4K77 peptide (should not block signal)

  • Peptides with other modifications at K77 (acetylation, etc.)

Dot blot specificity testing:

• Spot various synthetic peptides with different modifications on membrane

• Probe with antibody and quantify signal strength across modifications

• Create a cross-reactivity profile as a percentage of primary target reactivity

Western blot controls:

• Analyze samples with enzymatically removed modifications

• Include samples from cells treated with metabolic inhibitors that affect specific modifications

• Test recombinant histones with defined modification states

This approach is supported by studies that have used similar methods to evaluate the specificity of histone modification antibodies .

How can 2-hydroxyisobutyryl-HIST1H4A (K77) antibody be applied in multi-omic studies?

Multi-omic approaches integrating 2-hydroxyisobutyryl-HIST1H4A (K77) ChIP data with other datasets can provide comprehensive insights:

Integration with transcriptomics:

• Correlate H4K77 2-hydroxyisobutyrylation patterns with gene expression data

• Identify gene clusters with specific relationships between this modification and transcriptional activity

• Analyze cell type-specific patterns of correlation

Metabolomic integration:

• Investigate relationships between cellular metabolic state and 2-hydroxyisobutyrylation patterns

• Explore how metabolic perturbations affect the distribution of this modification

• Examine metabolic pathways that may influence 2-hydroxyisobutyryl-CoA availability

Proteomics approaches:

• Identify reader proteins that specifically recognize 2-hydroxyisobutyryl-HIST1H4A (K77)

• Map interaction networks connected to this modification

• Characterize enzymes responsible for writing or erasing this mark

Since histone modifications play a central role in transcription regulation, DNA repair, and replication , integrating these datasets can reveal functional relationships between 2-hydroxyisobutyryl-HIST1H4A (K77) and various cellular processes.

What considerations are important when using 2-hydroxyisobutyryl-HIST1H4A (K77) antibody in different cell types or tissues?

When applying the 2-hydroxyisobutyryl-HIST1H4A (K77) antibody across different biological systems, researchers should consider:

Cell type-specific variation:

• Baseline levels of 2-hydroxyisobutyrylation may vary significantly between cell types

• The functional significance of the modification may differ in specialized cells

• Extraction protocols may need optimization for specific tissues

Developmental context:

• The pattern and abundance of 2-hydroxyisobutyrylation may change during development

• Consider time-course experiments during differentiation or embryonic development

• Compare patterns between progenitor and terminally differentiated cells

Disease relevance:

• Evaluate changes in 2-hydroxyisobutyrylation patterns in disease states

• Compare normal and pathological tissues

• Consider pharmacological interventions that might affect this modification

Species-specific considerations:

• While the antibody is validated for human samples , cross-reactivity with other species should be empirically determined

• Histone H4 sequences are highly conserved, but modification patterns may differ between species

• Include appropriate controls when working with non-human samples