HIST1H3A (Ab-10) Antibody

What is HIST1H3A and what does the (Ab-10) antibody specifically recognize?

HIST1H3A encodes histone H3.1, one of the main histones responsible for nucleosome structure in eukaryotic chromosomal fibers. Histones are small, highly basic proteins consisting of a globular domain with unstructured N- and C-terminal tails that protrude from the main structure . The HIST1H3A (Ab-10) antibody specifically recognizes the region around serine 10 (Ser10) in the N-terminal tail of human histone H3.1 .

This antibody is derived from a peptide sequence surrounding the Ser10 site, which is a critical residue that undergoes post-translational modification, particularly phosphorylation during cell cycle progression . The specificity for this region makes this antibody valuable for studying histone modifications that affect or are affected by Ser10 status. Histone H3 has an observed molecular weight of approximately 15 kDa, although it typically migrates at around 17 kDa on SDS-PAGE gels .

What applications has the HIST1H3A (Ab-10) antibody been validated for?

The HIST1H3A (Ab-10) antibody has been validated for multiple research applications as shown in the table below:

The antibody has been successfully used on various sample types including HEK293 cell acid extracts and Jurkat cell acid extracts for Western blot applications . For immunohistochemistry, it has been validated on paraffin-embedded human colon cancer and lung cancer tissues . Immunofluorescence analysis has been successfully conducted in HeLa cells . When designing experiments, researchers should optimize these dilution ranges for their specific sample types and experimental conditions.

What is the reactivity profile of HIST1H3A (Ab-10) antibody?

The HIST1H3A (Ab-10) antibody is primarily reactive with human samples . This specificity is important when designing experiments with multiple species or when considering cross-species applications. The antibody is generated in rabbits as a polyclonal IgG isotype .

Unlike some other histone H3 antibodies which demonstrate broader species reactivity such as those that react with Arabidopsis thaliana, Botrytis cinerea, Chlamydomonas reinhardtii, and various plant species , the HIST1H3A (Ab-10) antibody is more species-targeted. This specificity is advantageous for human cell-based studies where cross-reactivity with non-human proteins could complicate data interpretation.

What are the optimal storage conditions for maintaining HIST1H3A (Ab-10) antibody activity?

For optimal maintenance of antibody activity, HIST1H3A (Ab-10) antibody should be stored according to the following guidelines:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Buffer composition: The antibody is typically supplied in a buffer containing 50% glycerol and 0.03% Proclin 300 as a preservative

Creating multiple small aliquots upon initial thawing is crucial to prevent repeated freeze-thaw cycles, which can degrade the antibody and reduce its effectiveness. Each aliquot should be sized appropriately for single-use applications to minimize waste and maintain consistency between experiments. The expected shelf life when stored properly is approximately 12 months from the date of receipt .

How should sample preparation be optimized for HIST1H3A (Ab-10) antibody use?

Sample preparation techniques vary by application, but there are several critical considerations for optimal results with HIST1H3A (Ab-10) antibody:

For Western blot analysis, acid extraction of histones is recommended for enrichment of histone proteins. This has been validated using Jurkat and HEK293 cell acid extracts . The antibody typically detects a band at approximately 17 kDa, corresponding to histone H3.1 .

For immunohistochemistry of paraffin-embedded tissues, antigen retrieval may be necessary. Based on similar histone H3 antibodies, antigen retrieval with TE buffer pH 9.0 is often suggested, though citrate buffer pH 6.0 may be used as an alternative . This step is crucial as formalin fixation can mask epitopes, particularly in the histone tail regions where post-translational modifications occur.

For ChIP applications, cells (approximately 4×10^6) should be treated with Micrococcal Nuclease followed by sonication before immunoprecipitation with 5 μg of the antibody . Including appropriate controls, such as normal rabbit IgG, is essential for validating specificity of chromatin enrichment.

How can potential cross-reactivity issues with histone modification antibodies be addressed in experimental design?

Cross-reactivity is a significant concern with histone antibodies due to the highly conserved nature of histone proteins and the presence of similar modification sites. HIST1H3A (Ab-10) antibody specifically targets the region around Ser10 of histone H3.1 , but there are important considerations for ensuring specificity.

Studies have shown that antibody recognition can be influenced by neighboring modifications. For example, some H3K9me3 antibodies are sensitive to neighboring H3S10 phosphorylation, which can lead to under-representation of singly-marked histone H3 populations, particularly during mitosis . To address this, researchers should:

• Include appropriate controls in each experiment, including a non-specific IgG control and, when possible, samples lacking the target modification

• Validate findings using multiple antibodies that recognize the same modification but were raised against different epitopes

• Consider performing peptide competition assays with modified and unmodified peptides to confirm specificity

• For critical experiments, validate results with alternative techniques such as mass spectrometry

In ChIP experiments, researchers should perform parallel ChIP-Seq in genetic knockout or knockdown models where possible. For example, studies have validated H3K27 methylation antibodies by performing parallel ChIP-Seq in cells lacking H3K27 methylation due to genetic deletion of the PRC2 complex . This approach provides definitive evidence of antibody specificity.

What are the technical considerations for using HIST1H3A (Ab-10) antibody in cell cycle studies?

The Ser10 region recognized by HIST1H3A (Ab-10) antibody is particularly important in cell cycle regulation, as H3S10 phosphorylation is dynamically regulated during cell cycle progression. This modification is found in low abundance at transcriptionally active genes in interphase cells but becomes highly enriched in mitotic chromatin during condensation .

For effective cell cycle studies using this antibody:

• Cell synchronization: Methods such as double thymidine block to arrest cells at G1/S can be employed, followed by timecourse analysis after release. A previous study using H3S10p antibodies showed that signal was weak through S phase and pronounced in mitotic extracts .

• Co-staining approaches: Combine HIST1H3A (Ab-10) antibody with markers of different cell cycle phases (such as cyclin antibodies or DNA content staining) for more precise correlation of modifications with cell cycle stage.

• Time-resolved analysis: For investigating dynamic changes, collect samples at regular intervals following synchronization release (typically every 2-3 hours for a complete cell cycle).

• Quantitative analysis: For immunofluorescence, quantify signal intensity across different cell cycle stages using image analysis software. This provides more objective data than qualitative assessment.

• Controls: Include both positive controls (mitotic cells for H3S10p) and negative controls (interphase cells or phosphatase-treated samples).

The combination of these approaches allows for robust analysis of histone modifications throughout the cell cycle while minimizing artifacts that could arise from single-technique approaches.

How does phosphorylation at Ser10 affect histone H3 antibody recognition and what technical approaches can address this?

Phosphorylation at Ser10 of histone H3 significantly impacts antibody recognition of neighboring modifications, creating a critical technical consideration for experimental design. Research has shown that H3K9me3 antibodies can be sensitive to neighboring H3S10 phosphorylation, potentially leading to under-representation of certain modified histone populations, especially during mitosis when H3S10 phosphorylation is abundant .

To address these technical challenges:

• Use modification-specific antibodies with known behavior around Ser10: Some H3K9me3 antibodies (such as the referenced Ab2) are insensitive to neighboring H3S10p, making them suitable for detecting this modification regardless of phosphorylation status .

• Employ combinatorial approaches: Use combinations of selective reagents to accurately detect and map dually marked chromatin signatures. For example, when studying H3K9me3 during mitosis, use antibodies verified to be insensitive to H3S10p status.

• Validate with phosphatase treatment: For crucial experiments, compare antibody binding in paired samples with and without phosphatase treatment to determine how phosphorylation affects epitope recognition.

• Western blot analysis of synchronized cells: Track the cell cycle dynamics of histone modifications using synchronized cells and compare the results with multiple antibodies that recognize the same modification but differ in sensitivity to neighboring modifications .

• Consider sequential ChIP: For investigating co-occurrence of modifications, sequential ChIP (re-ChIP) with different modification-specific antibodies can provide evidence of true bivalent domains.

These approaches help ensure accurate interpretation of data involving histone modifications around the Ser10 site, particularly in contexts where phosphorylation levels change dynamically.

What validation strategies should be employed when using HIST1H3A (Ab-10) antibody in new experimental systems?

When introducing HIST1H3A (Ab-10) antibody to new experimental systems, comprehensive validation is essential to ensure reliable and interpretable results. The following strategies are recommended:

• Multi-technique validation: Confirm antibody performance across multiple techniques (WB, IF, IHC, ChIP) to establish consistent recognition of the target.

• Peptide competition assays: Perform binding competition with both phosphorylated and non-phosphorylated peptides corresponding to the H3 Ser10 region to confirm specificity for the intended modification state.

• Genetic models: When possible, utilize genetic models with altered H3S10 phosphorylation levels, such as Aurora kinase inhibition or knockout models, to confirm specificity.

• Positive and negative controls: Include samples with known high levels of H3S10 phosphorylation (e.g., mitotic cells) and samples where this modification is absent or reduced.

• Quantitative assessment: For ChIP experiments, perform quantitative PCR analysis of immunoprecipitated DNA using primers targeting regions known to be enriched or depleted for the modification of interest .

• Cross-validation with other antibodies: Compare results with other established antibodies targeting the same region or modification to identify any discrepancies that may indicate technical issues.

• Signal-to-noise optimization: Titrate antibody concentrations to determine the optimal dilution that maximizes specific signal while minimizing background. The recommended ranges (WB:1:500-5000, IHC-P:1:20-200, IF:1:50-200) should be optimized for each system .

These validation approaches ensure that findings are robust and reproducible, particularly when extending the use of the antibody to new cell types, tissues, or experimental conditions.

What are the technical considerations for optimizing ChIP protocols with HIST1H3A (Ab-10) antibody?

Chromatin Immunoprecipitation (ChIP) with HIST1H3A (Ab-10) antibody requires careful optimization for successful results. Based on validated protocols, the following technical considerations are critical:

• Chromatin preparation:

  • Treat approximately 4×10^6 cells with Micrococcal Nuclease followed by sonication to generate appropriately sized chromatin fragments (typically 200-500 bp)

  • Over-sonication can destroy epitopes, while insufficient fragmentation reduces IP efficiency

  • Optimize sonication conditions for each cell type

• Antibody amount:

  • Use 5 μg of HIST1H3A (Ab-10) antibody per ChIP reaction with 4×10^6 cells

  • Include a matched non-specific IgG control at the same concentration

• Quantification methods:

  • Quantify immunoprecipitated DNA using real-time PCR with primers for regions known to be enriched for the target modification

  • For genome-wide studies, perform ChIP-Seq with appropriate sequencing depth (minimum 20 million reads recommended)

• Controls and validation:

  • For critical experiments, perform parallel ChIP-Seq in cell lines lacking the modification through genetic or chemical means

  • Meta-analysis of average signal over known peaks can confirm antibody specificity

• Cross-linking conditions:

  • Formaldehyde cross-linking time should be optimized (typically 10-15 minutes)

  • Over-fixation can mask epitopes, particularly in histone tails

• Washing stringency:

  • Balance between removing non-specific interactions and maintaining specific binding

  • Consider testing different salt concentrations in wash buffers

• Data analysis considerations:

  • For ChIP-Seq, focus analysis on unique peaks that are absent in IgG control samples

  • Compare signal distribution to known patterns of H3S10 phosphorylation

Optimization of these parameters for each experimental system is crucial for obtaining reliable and reproducible ChIP results with HIST1H3A (Ab-10) antibody.