2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody

What is 2-hydroxyisobutyrylation and why is it significant in epigenetic research?

2-hydroxyisobutyrylation is a relatively newly discovered histone post-translational modification that plays critical roles in chromatin remodeling and gene transcription regulation. This modification occurs on lysine residues of histone proteins, including HIST1H2BC. It represents an important epigenetic mark that influences DNA accessibility and transcriptional activity by altering chromatin structure . Unlike better-studied modifications such as acetylation or methylation, 2-hydroxyisobutyrylation provides unique insights into specialized regulatory mechanisms of gene expression. The study of this modification is particularly valuable for understanding cell-type specific gene regulation and responses to environmental factors that may influence disease states .

How does the 2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody differ from other site-specific antibodies?

The 2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody specifically recognizes the 2-hydroxyisobutyryl modification at the K24 residue of the HIST1H2BC protein. This site-specificity distinguishes it from other antibodies that target different lysine residues on the same histone variant. For comparison, other antibodies in the same family target K12, K34, or K120 sites, each associated with distinct regulatory functions . The K24 site has a unique position within the histone tail that may affect different protein-protein interactions or chromatin structures compared to modifications at other sites. Using multiple site-specific antibodies in parallel experiments can provide a comprehensive view of how different modifications on the same histone protein coordinate to regulate gene expression.

What are the validated applications for this antibody?

Based on data from related antibodies in the same family, the 2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody is likely validated for multiple experimental applications including:

Similar to other antibodies in this family, this antibody is most likely reactive with human samples and requires careful optimization for each specific application .

What is the recommended protocol for Western blotting with this antibody?

For optimal Western blotting results with the 2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody, follow this detailed protocol:

• Extract proteins using RIPA buffer followed by brief sonication to ensure complete histone extraction

• Determine protein concentration using BCA assay

• Load 10-20 μg of protein per lane on a 15% SDS-PAGE gel (optimal for histone separation)

• Transfer proteins to PVDF membrane (preferred over nitrocellulose for histone proteins)

• Block membrane with Odyssey Blocking Buffer for 1.5 hours at room temperature

• Incubate with primary antibody at 1:500 dilution overnight at 4°C

• Wash 3 times with TBST (TBS + 0.1% Tween-20)

• Incubate with appropriate secondary antibody (IR dye-conjugated recommended for quantitative analysis)

• Visualize using an infrared imaging system for optimal quantification

For enhanced detection of the 2-hydroxyisobutyrylation signal, pre-treatment of cells with 30mM sodium butyrate for 4 hours before lysis can increase the modification levels, as demonstrated with other site-specific antibodies in this family .

How can background signals be reduced in Western blot experiments?

High background is a common challenge when working with histone modification antibodies. To minimize background:

• Increase blocking time to 2 hours using 5% BSA in TBST instead of standard blocking buffers

• Ensure complete removal of SDS from the gel by extending transfer time

• Use freshly prepared buffers for all steps

• Increase washing duration and frequency (5 washes of 5 minutes each)

• Optimize primary antibody concentration through titration experiments

• Consider using detergent additives such as 0.1% Triton X-100 in antibody dilution buffers

• Pre-absorb the antibody with non-specific proteins if cross-reactivity is observed

Background issues can also indicate sample quality problems. Ensure histone proteins are properly extracted and not degraded by using fresh samples and appropriate protease inhibitors in all buffers.

What controls should be included in experiments with this antibody?

Proper experimental controls are essential for interpreting results with the 2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody:

For the knockdown validation, consider generating stable HIST1H2BC knockdown cell lines using lentiviral particles containing small hairpin RNA as described in the literature . This provides the most stringent test of antibody specificity.

How can this antibody be used in chromatin immunoprecipitation (ChIP) experiments?

While not explicitly validated for ChIP in the provided information, based on properties of similar histone modification antibodies, the following protocol is recommended:

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

• Quench with 0.125M glycine for 5 minutes

• Lyse cells and isolate nuclei

• Sonicate chromatin to fragments of 200-500bp

• Pre-clear chromatin with protein A/G beads

• Immunoprecipitate with 5μg of 2-hydroxyisobutyryl-HIST1H2BC (K24) Antibody overnight at 4°C

• Capture antibody-chromatin complexes with protein A/G beads

• Wash extensively with increasing stringency buffers

• Elute and reverse cross-links

• Purify DNA for qPCR or sequencing analysis

For ChIP-seq applications, ensure antibody has high specificity and low background by performing preliminary ChIP-qPCR validation at known target regions before proceeding to genome-wide analysis.

What is the significance of studying HIST1H2BC K24 2-hydroxyisobutyrylation in cancer research?

HIST1H2BC modification patterns have been implicated in various cancers, making the study of site-specific modifications particularly relevant. Research indicates that histone H2B variants, including HIST1H2BC, play roles in endocrine-resistant breast cancer . The 2-hydroxyisobutyryl modification likely contributes to altered gene expression programs in cancer cells through several mechanisms:

• Changed chromatin accessibility at cancer-related gene promoters

• Altered recruitment of transcription factors and chromatin remodelers

• Disruption of normal cell cycle regulation

• Modified response to hormone treatment in hormone-dependent cancers

Studies using HIST1H2BC overexpression in MCF-7 cells showed 10-13 fold increases, suggesting this histone variant's importance in breast cancer biology . The K24 modification site may provide a specific regulatory node that could be targeted therapeutically if its role in oncogenic pathways is established.

How does 2-hydroxyisobutyrylation at K24 compare with other modifications on HIST1H2BC?

The HIST1H2BC protein undergoes various modifications at different lysine residues, each with potential distinct functions:

The functional differences between these sites highlight the complexity of the histone code. The K24 site's proximity to K12 suggests possible coordination between these modifications, potentially creating combinatorial effects on gene regulation . Experimental approaches combining multiple site-specific antibodies can elucidate how these modifications work in concert to regulate gene expression programs.

What techniques can provide quantitative measurement of global 2-hydroxyisobutyrylation levels?

For quantitative assessment of global 2-hydroxyisobutyrylation levels, researchers should consider:

• Mass spectrometry-based approaches

  • Provides precise quantification of modification levels

  • Can identify multiple modifications simultaneously

  • Requires specialized equipment and expertise

• ELISA-based quantification

  • Use antibody at 1:2000-1:10000 dilution

  • Develop standard curves with modified peptides

  • Suitable for high-throughput screening

• Western blot densitometry

  • Semi-quantitative approach

  • Normalize to total H2B levels

  • Use infrared detection systems for better quantification

These techniques can be particularly valuable when studying how environmental factors or drug treatments affect global histone modification patterns.