Acetyl-HIST1H3A (K27) Antibody

What are the recommended applications for Acetyl-HIST1H3A (K27) antibody?

Acetyl-HIST1H3A (K27) antibodies have been validated for multiple applications with specific dilution recommendations:

How should I store and handle Acetyl-HIST1H3A (K27) antibody?

Proper storage significantly impacts antibody performance:

• Store at -20°C for long-term or -80°C for extended preservation

• Aliquot upon receipt to avoid repeated freeze-thaw cycles

• Most formulations contain 50% glycerol with preservatives like 0.03% Proclin 300

• Working dilutions can be stored at 4°C for short-term use (1-2 weeks)

• After thawing, centrifuge briefly before opening to collect all material at the bottom of the tube

What controls should I include when using this antibody?

Controls are essential for interpreting H3K27ac experimental results:

• Positive control: HeLa cells treated with HDAC inhibitors (sodium butyrate at 30mM for 4h or Trichostatin A at 500ng/ml for 4h)

• Negative control: IgG from same species as the primary antibody

• Peptide competition: Pre-incubating antibody with acetylated H3K27 peptide should abolish signal

• Cell type controls: MCF-7, Jurkat, and A549 cells show variable baseline H3K27ac levels

Include at least one non-histone protein control (e.g., β-actin) to normalize for loading variations.

How can I distinguish between H3K27 acetylation and other histone modifications?

Cross-reactivity is a critical concern in histone modification research:

• Use antibodies with verified specificity like EP16602 clone, which binds K27ac alone and when S28 is phosphorylated

• Perform peptide array tests to confirm specificity against other acetylated lysine residues (K9, K14, K18, K23)

• For simultaneous detection of multiple modifications, use sequential ChIP or co-immunoprecipitation approaches

• Validate results with at least two different antibody clones or techniques (WB and ChIP-seq)

Data from peptide competition assays demonstrate that high-quality antibodies show >95% reduction in signal when pre-incubated with specific acetylated peptides but maintain signal with non-acetylated or differently acetylated peptides .

What factors affect the detection sensitivity in ChIP experiments?

ChIP optimization for H3K27ac requires careful consideration of multiple variables:

• Crosslinking time: Excessive crosslinking can mask the H3K27ac epitope; limit to 10 minutes with 1% formaldehyde

• Sonication conditions: Aim for chromatin fragments between 200-500bp for optimal H3K27ac detection

• Antibody amount: Use 5μg antibody for 5-10μg of chromatin for best signal-to-noise ratio

• Washing stringency: Higher salt concentrations in wash buffers reduce background but may decrease specific signal

• Cell number: Starting with at least 1×10^6 cells ensures sufficient material for H3K27ac ChIP

In ChIP-seq applications, the EP16602 and RM172 clones have demonstrated superior enrichment profiles at active enhancer and promoter regions compared to polyclonal alternatives .

How do H3K27 mutations affect antibody binding and cell biology?

Research examining H3K27 mutations reveals important biological implications:

• K27M and K27I mutations in H3.1 variants expand hematopoietic stem cell (HSC) populations in vivo

• Mutated HSCs showed significantly higher engraftment (average 30%) compared to wildtype controls (avg. 0.09%)

• These mutations may interfere with antibody binding by preventing K27 acetylation

• H3K27 mutations are early events in leukemogenesis and drive pre-cancerous stem cell expansion

This data demonstrates the profound biological effects of K27 mutations and underscores the importance of acetylation at this position .

What is the optimal protocol for Western blot detection of H3K27ac?

For reproducible Western blot results with H3K27ac antibodies:

• Extract histones using specialized acid extraction methods to enrich for nuclear proteins

• Add histone deacetylase inhibitors (e.g., sodium butyrate) to lysis buffers

• Use SDS-PAGE gels with higher percentages (15-18%) for better separation of histone proteins

• Transfer to PVDF membranes at lower voltage (30V) overnight for efficient transfer of small histone proteins

• Block with 5% BSA rather than milk (milk contains biotin that can cause background)

• Dilute primary antibody 1:1000 in 5% BSA/TBST and incubate overnight at 4°C

• Use HRP-conjugated secondary antibodies at 1:40000 dilution for reduced background

Most studies report a specific band at approximately 17kDa corresponding to acetylated H3K27 .

How should I interpret ChIP-seq data generated with H3K27ac antibodies?

ChIP-seq analysis requires specialized analytical approaches:

• H3K27ac marks are predominantly found at active enhancers and promoters

• Use appropriate peak-calling algorithms optimized for histone modifications (e.g., MACS2 with broad peak options)

• Differentiate between promoter-associated (TSS-proximal) and enhancer-associated (distal) H3K27ac peaks

• Compare with other histone modifications (H3K4me1, H3K4me3) to distinguish enhancers from promoters

• Assess signal distribution patterns; typical H3K27ac peaks show broader distribution than transcription factor binding sites

• Normalize to input controls and total H3 occupancy for accurate quantification

The EP16602 clone has been specifically validated for ChIP-seq applications and shows robust enrichment profiles at known regulatory elements .

Why might I observe non-specific bands in Western blot?

Non-specific bands are a common challenge with histone antibodies:

• Cause: Incomplete blocking, antibody concentration too high, or cross-reactivity with other acetylated histones

• Solution: Increase blocking time to 2 hours with 5% BSA/TBST

• Cause: Sample degradation or histone deacetylase activity

• Solution: Add protease inhibitors and HDAC inhibitors (sodium butyrate) to extraction buffers

• Cause: Loading too much protein

• Solution: Perform titration experiments with 5-20μg total protein to determine optimal loading

Pre-adsorbing the antibody with acetylated peptides other than H3K27ac can help identify if cross-reactivity is causing non-specific bands .

How can I improve signal intensity in immunofluorescence experiments?

For stronger and more specific immunofluorescence signals:

• Optimize cell fixation: 10-minute fixation with 4% paraformaldehyde preserves H3K27ac epitopes better than methanol

• Include permeabilization step with 0.2% Triton X-100 for 10 minutes to improve nuclear accessibility

• Block with 10% normal goat serum for at least 30 minutes at room temperature

• Use higher antibody concentrations (1:5 to 1:10 dilution) for immunofluorescence than for Western blot

• Extend primary antibody incubation to overnight at 4°C

• Use signal amplification systems like biotin-streptavidin or tyramide signal amplification for weak signals

• Counterstain with DAPI to visualize nuclei and confirm nuclear localization of H3K27ac signal

Treatments that increase H3K27 acetylation (sodium butyrate 30mM for 4h) provide excellent positive controls for IF optimization .

What is the functional significance of H3K27 acetylation in gene regulation?

H3K27 acetylation plays critical roles in regulating gene expression:

• Marks active enhancers and promoters, often co-occurring with H3K4me1 at enhancers

• Antagonizes Polycomb-mediated H3K27 trimethylation (H3K27me3), a repressive mark

• Facilitates chromatin accessibility by disrupting histone-DNA interactions

• Serves as a binding platform for bromodomain-containing proteins like BRD4

• Dynamically regulated by histone acetyltransferases (p300/CBP) and deacetylases (HDACs)

Studies examining H3K27 mutations in histones demonstrate their role in driving hematopoietic stem cell expansion, highlighting the importance of this modification in controlling cell fate and differentiation .

How does H3K27ac distribution change during cellular differentiation?

Understanding H3K27ac dynamics during differentiation requires specialized approaches:

• Time-course ChIP-seq experiments at critical differentiation timepoints

• Integration with transcriptome data (RNA-seq) to correlate H3K27ac changes with gene expression

• Analysis of cell-type-specific enhancers marked by H3K27ac

• Comparison between progenitor and differentiated cell populations

• Identification of pioneer factors that establish H3K27ac at lineage-specific enhancers

Research on hematopoietic stem cells has shown that H3K27 modifications influence engraftment potential and differentiation capacity, with specific mutations expanding the stem cell compartment .