Histone H3 (Ab-27) Antibody

What is Histone H3 (Ab-27) Antibody and what epitope does it specifically recognize?

Histone H3 (Ab-27) Antibody is a rabbit polyclonal antibody that recognizes the peptide sequence around amino acids 26-30 (A-R-K-S-A) derived from human Histone H3. This antibody detects endogenous levels of total Histone H3 protein rather than specific modifications. The antibody is produced by immunizing rabbits with synthetic peptide and KLH conjugates, followed by purification through affinity-chromatography using epitope-specific peptide . Unlike modification-specific antibodies, Histone H3 (Ab-27) Antibody recognizes the core region of histone H3, making it valuable for normalizing histone levels in epigenetic studies regardless of post-translational modifications.

How does Histone H3 (Ab-27) Antibody reactivity compare across different species?

Histone H3 (Ab-27) Antibody demonstrates confirmed reactivity with human, mouse, and rat samples, as indicated by the product specifications . This cross-species reactivity is expected due to the highly conserved nature of histone H3 across different species. For comparison, other histone H3 antibodies like the one described in search result show confirmed reactivity with a broader range of species including Arabidopsis thaliana, Botrytis cinerea, Chlamydomonas reinhardtii, Hordeum vulgare, Phaeodactylum tricornutum, Physcomitrium patens, and Zea mays, reflecting the evolutionary conservation of histone H3 sequences . When using Histone H3 (Ab-27) Antibody with species not explicitly listed in its reactivity profile, validation experiments should be conducted to confirm specificity.

What is the optimal protocol for using Histone H3 (Ab-27) Antibody in Western blotting experiments?

For optimal Western blotting results with Histone H3 (Ab-27) Antibody, researchers should follow this methodological approach:

• Sample preparation: Extract histones using acid extraction methods (0.2N HCl or sulfuric acid extraction), as histone proteins are relatively small (17 kDa for H3) and highly basic.

• Gel selection: Use 15-18% SDS-PAGE gels to provide optimal separation of histone proteins.

• Dilution optimization: The recommended starting dilution is 1:5000, though this may require adjustment based on sample type and detection system .

• Optimization considerations: For challenging samples, consider the 1% SDS hot lysis method as recommended for similar histone H3 antibodies .

• Detection: Use appropriate secondary antibodies (anti-rabbit IgG conjugated to HRP) and ECL detection systems.

• Expected results: A band at approximately 17 kDa should be detected, corresponding to histone H3 .

Proper sample preparation is critical because histones are tightly bound to DNA in chromatin. Inclusion of appropriate controls, such as recombinant histone H3 or previously validated cell/tissue extracts, should be incorporated to ensure specificity.

How can Histone H3 (Ab-27) Antibody be effectively used in chromatin immunoprecipitation (ChIP) experiments?

While Histone H3 (Ab-27) Antibody is primarily recommended for Western blotting applications , it can be adapted for ChIP experiments following these methodological considerations:

• Crosslinking optimization: Standard formaldehyde crosslinking (1% for 10 minutes at room temperature) is typically sufficient for histone H3 ChIP experiments.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp.

• Antibody amount: For ChIP experiments with total H3 antibodies, a recommended starting point is 2-5 μg antibody per IP reaction with 10-25 μg of chromatin (similar to protocols used with comparable antibodies) .

• Controls: Include IgG negative controls and positive controls targeting known abundant histone marks.

• Data normalization: Total H3 ChIP data can serve as a normalization control for histone modification ChIP experiments (e.g., H3K27me3, H3K27ac) to account for nucleosome density variations.

For validation, researchers should target genomic regions known to contain nucleosomes, such as actively transcribed genes (for positive signals) and gene deserts (for background assessment).

How can researchers differentiate between specificity issues and technical artifacts when using Histone H3 (Ab-27) Antibody?

When troubleshooting experiments with Histone H3 (Ab-27) Antibody, researchers should implement the following methodological approach:

• Epitope masking assessment: Determine if post-translational modifications near the antibody epitope (aa.26-30) might be interfering with antibody binding. This region is close to K27, which can be acetylated or methylated, potentially affecting antibody recognition .

• Cross-reactivity validation: Implement peptide competition assays using the immunizing peptide to confirm binding specificity.

• Sample preparation validation: Compare different extraction methods (e.g., acid extraction vs. RIPA buffer) as some methods may better preserve the epitope structure. Evidence from similar antibodies shows that the extraction method can significantly impact detection efficiency .

• Antibody dilution optimization: Test a range of antibody dilutions (1:1000-1:10000) to establish the optimal signal-to-noise ratio.

• Lot-to-lot variation assessment: When switching to a new lot, perform side-by-side comparisons with the previous lot to detect potential manufacturing variations.

Research has shown that even well-characterized histone antibodies can exhibit unexpected cross-reactivity. For example, search result describes a case where an H3K27me3 antibody showed cross-reactivity with H3K4me3-marked histones in cells, presenting potential challenges for bivalent chromatin studies .

What methodologies can address potential cross-reactivity between Histone H3 (Ab-27) Antibody and modified forms of H3?

To address potential cross-reactivity issues, researchers should implement these methodological approaches:

• Peptide array validation: Utilize peptide arrays containing various modified histone peptides to assess potential cross-reactivity, similar to approaches described for other histone antibodies . These arrays typically include unmodified H3 peptides and H3 peptides with various modifications at different residues.

• Knockout/knockdown controls: When possible, use samples from systems where specific histone-modifying enzymes have been depleted, similar to the SET1 deletion approach mentioned in search result .

• Competitive ELISA: Implement competitive ELISA assays using modified and unmodified peptides to quantitatively assess antibody specificity.

• Sequential ChIP: For ChIP applications, sequential ChIP with antibodies against specific modifications followed by Histone H3 (Ab-27) Antibody can help determine if the same nucleosomes are being recognized.

Data from research on similar antibodies suggests that cross-reactivity between different histone modifications can occur. For example, antibodies against H3K27me3 have shown weak binding (14%) to H3K27me2 modifications in ELISA tests , highlighting the importance of rigorous specificity validation.

How can Histone H3 (Ab-27) Antibody be effectively used as a normalization control in studies of histone modifications at lysine 27?

For studies focusing on H3K27 modifications, Histone H3 (Ab-27) Antibody can serve as an essential normalization control through the following methodological approach:

• Parallel sample processing: Process identical aliquots of samples for both total H3 and specific H3K27 modification detection (H3K27me3, H3K27ac).

• Quantitative analysis: When analyzing Western blot data, calculate the ratio of modification signal to total H3 signal to account for variations in histone loading or extraction efficiency.

• ChIP-seq normalization: In ChIP-seq studies, perform parallel ChIP with Histone H3 (Ab-27) Antibody and modification-specific antibodies, then normalize modification enrichment to total H3 occupancy at each genomic region.

• Multi-antibody validation: Include antibodies against multiple distinct H3K27 modifications (H3K27me3, H3K27ac) to comprehensively assess the chromatin state, as these modifications are mutually exclusive and associated with opposing transcriptional states .

Research has demonstrated that H3K27 modifications play critical and opposing roles in gene regulation: H3K27 acetylation is associated with active transcription and enhancer activation, while H3K27 trimethylation is associated with gene silencing by Polycomb group proteins . Proper normalization to total H3 levels is essential for accurate interpretation of these modification patterns.

What methodological considerations are necessary when using Histone H3 (Ab-27) Antibody in studies of cancer epigenetics?

When applying Histone H3 (Ab-27) Antibody in cancer epigenetics research, these methodological considerations are critical:

• Heterogeneity assessment: Cancer tissues exhibit cellular heterogeneity, requiring careful microdissection or single-cell approaches to obtain accurate histone profiles.

• Comparative analysis: Always include matched normal tissues for comparison, as alterations in total histone H3 levels may occur in cancer tissues.

• Multi-technique validation: Combine Western blotting with immunohistochemistry to correlate biochemical data with spatial distribution in tumor sections.

• Normalization strategy: Consider using multiple normalization controls beyond traditional housekeeping proteins, as cancer cells often exhibit altered expression of these proteins.

• Quantitative assessment: Implement densitometric analysis of Western blots to precisely quantify differences between normal and tumor samples.

Research has shown that histone H3 modifications at K27 are altered in colorectal cancer samples. Data from search result demonstrates that both H3K27 trimethylation and H3K27 acetylation show statistically significant increases in colorectal cancer compared to normal mucosa (fold changes of 1.67 and 1.54, respectively, p < 0.05) . These findings highlight the importance of assessing both total H3 levels and specific modifications in cancer studies.

Table 1: Alterations in histone modifications in colorectal cancer (CRC) compared to normal colon (NC). FC: fold change.

How might Histone H3 (Ab-27) Antibody be incorporated into emerging chromatin profiling techniques like CUT&RUN and CUT&Tag?

While Histone H3 (Ab-27) Antibody is primarily validated for Western blotting, its potential application in emerging techniques like CUT&RUN and CUT&Tag should follow these methodological considerations:

• Antibody validation: Before implementing in these techniques, validate the antibody specifically for CUT&RUN/CUT&Tag using positive controls and spike-in normalization.

• Protocol adaptation: CUT&RUN and CUT&Tag protocols typically use 0.5-2 μg of antibody per reaction, which may need optimization for Histone H3 (Ab-27) Antibody.

• Spike-in controls: Incorporate spike-in controls like those mentioned in search result (SNAP-CUTANA Spike-in Controls) to ensure proper normalization and quantification.

• Comparative assessment: Run parallel reactions with established CUT&RUN/CUT&Tag-validated H3 antibodies to benchmark performance.

• Data analysis optimization: Implement specialized peak calling parameters suitable for broadly distributed histone marks like total H3.

These emerging techniques offer advantages over traditional ChIP by requiring fewer cells and providing improved signal-to-noise ratios. Research using similar antibodies like the H3K27me3 SNAP-Certified antibody described in search result demonstrates the effectiveness of these techniques for histone profiling when properly optimized.

What role can Histone H3 (Ab-27) Antibody play in understanding the relationship between histone H3 variants and their modifications?

To investigate histone H3 variants and their modifications, researchers can apply Histone H3 (Ab-27) Antibody with these methodological approaches:

• Variant-specific comparison: Use in parallel with variant-specific antibodies (H3.1, H3.2, H3.3) to assess the distribution of modifications across different H3 variants.

• Immunoprecipitation-mass spectrometry: Combine immunoprecipitation using Histone H3 (Ab-27) Antibody with mass spectrometry to identify both variants and their modifications simultaneously.

• Sequential ChIP: Perform sequential ChIP with variant-specific antibodies followed by Histone H3 (Ab-27) Antibody to determine the enrichment of specific variants at genomic regions.

• Developmental timing analysis: Assess temporal changes in H3 variant distribution during developmental processes or cellular differentiation.

Research has demonstrated that histone variants play crucial roles in gene regulation. For instance, search result describes how a non-modifiable variant of H3.3 at residue K27 (H3.3K27A) causes severe developmental defects in Arabidopsis, including early flowering, increased callus formation, and alterations in stem development and lignin biosynthesis . This highlights how specific residues in histone variants can serve as critical regulatory points for gene expression and development.

Research has also shown that H3.3 is preferentially incorporated into the male pronucleus after fertilization and acquires trimethylation at lysine 27 in mice, which is necessary for normal embryonic development . This example illustrates how histone variants and their modifications coordinate developmental processes and how antibodies recognizing core histone regions can help elucidate these mechanisms.

What are the optimal crosslinking and chromatin preparation methods when using Histone H3 (Ab-27) Antibody for ChIP applications?

For optimal chromatin preparation when using Histone H3 (Ab-27) Antibody in ChIP applications, researchers should implement the following methodological approach:

• Crosslinking optimization: For histone H3, standard formaldehyde crosslinking (1% for 10 minutes at room temperature) is typically effective, but dual crosslinking with formaldehyde followed by ethylene glycol bis(succinimidyl succinate) (EGS) can enhance recovery for some applications.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500 bp, which is ideal for histone ChIP experiments. Over-sonication can lead to epitope destruction, while under-sonication results in poor resolution.

• Enzymatic digestion alternative: Consider micrococcal nuclease (MNase) digestion instead of sonication, as it generates nucleosome-sized fragments (~150 bp) that are ideal for histone studies.

• Buffer optimization: Chromatin immunoprecipitation buffer components can significantly impact antibody performance. For Histone H3 antibodies, buffers containing 0.1% SDS, 1% Triton X-100, and 150-300 mM NaCl typically yield good results, similar to conditions used for related antibodies in search result .

• Fragment size verification: Verify chromatin fragmentation by analyzing a small aliquot of de-crosslinked DNA on an agarose gel before proceeding with immunoprecipitation.

Research protocols for similar histone H3 antibodies recommend using 20 μl of antibody and 10 μg of chromatin (approximately 4 x 10^6 cells) per IP reaction for optimal ChIP results . Proper optimization of these parameters is essential for generating reproducible and reliable data.

How can Histone H3 (Ab-27) Antibody be integrated into multi-omic studies investigating histone-DNA interactions?

For integration of Histone H3 (Ab-27) Antibody into multi-omic research frameworks, implement the following methodological approach:

• Parallel -omics workflow design: Design experiments that simultaneously analyze:

  • ChIP-seq (using Histone H3 and modification-specific antibodies)

  • RNA-seq (to correlate histone occupancy with gene expression)

  • ATAC-seq (to assess chromatin accessibility)

  • DNA methylation profiling (to investigate crosstalk between histone marks and DNA methylation)

• Sequential chromatin immunoprecipitation: Perform sequential ChIP with Histone H3 (Ab-27) Antibody and other antibodies targeting specific modifications to identify co-occurrence patterns.

• Integrated data analysis: Implement bioinformatic pipelines that can integrate multi-omic datasets to identify correlations between histone occupancy, modifications, and functional outcomes.

• Single-cell adaptation: Consider adapting protocols for single-cell applications to address cellular heterogeneity in complex tissues.

Research has demonstrated that histone H3 modifications operate independently of other epigenetic mechanisms in some contexts. For example, search result revealed that H3K27me3 in Arabidopsis is a major and systematic gene silencing mechanism that acts independently of small RNAs or DNA methylation . Such findings highlight the importance of integrated multi-omic approaches to fully understand the complex interplay between different epigenetic mechanisms.

What methodological considerations are important when using fluorescence polarization assays with histone H3 antibodies for binding studies?

For researchers implementing fluorescence polarization (FP) assays to study histone H3 binding interactions, the following methodological considerations are crucial:

• Instrument calibration: Calibrate fluorescence polarization readers using standardized calibration dyes to determine the G-factor and establish instrument sensitivity, as outlined in search result .

• Peptide concentration optimization: Determine the optimal final concentration of fluorescently labeled histone H3 peptides (typically 100-500 nM depending on instrument sensitivity) .

• Buffer composition standardization: Prepare appropriate binding buffers that maintain protein stability while minimizing background fluorescence. For histone studies, buffers typically contain:

  • 20 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 0.01% Triton X-100

  • 1 mM DTT (added fresh)

• Serial dilution strategy: Perform 1:2 serial dilutions of the binding protein (starting from ~5 μM) to generate accurate binding curves and determine dissociation constants (Kd).

• Data analysis optimization: Use appropriate curve-fitting software (such as Kaleidagraph, SigmaPlot, GraphPad Prism, or OriginPro) to accurately analyze binding data and calculate binding affinities .