HIST1H3A (Ab-28) Antibody

What is HIST1H3A and why is phosphorylation at serine 28 important?

HIST1H3A (Histone Cluster 1, H3a) is a variant of histone H3, one of the core histones that form the nucleosome structure in eukaryotic chromatin. Phosphorylation at serine 28 (pSer28) of histone H3 is a critical post-translational modification associated with chromosome condensation during mitosis and is also involved in gene regulation processes. This modification is mediated by kinases such as MSK1 following activation of the MAP kinase signaling pathway in response to stimuli like UV radiation, EGF, and oncoproteins like c-Myc, c-Jun, and c-Fos . Understanding H3 pSer28 is crucial for investigating cellular processes like cell division, transformation, and transcriptional regulation.

How do HIST1H3A (Ab-28) antibodies differ from other histone H3 antibodies?

HIST1H3A (Ab-28) antibodies specifically recognize histone H3 phosphorylated at serine 28, distinguishing them from antibodies targeting other H3 modifications such as phosphorylation at serine 10 (pSer10), various lysine methylations (e.g., H3K4me3, H3K9me3, H3K27me3), or acetylations (e.g., H3K9ac, H3K27ac). The HTA28 monoclonal antibody, for example, specifically recognizes phosphorylated serine 28 of human, mouse, rat, bovine, and hamster histone H3 without cross-reactivity to phosphorylated serine 10 . This specificity makes these antibodies valuable tools for distinguishing between different phosphorylation states that may have distinct biological functions.

What are the typical applications for HIST1H3A (Ab-28) antibodies?

HIST1H3A (Ab-28) antibodies are commonly used in:

• Flow cytometric analysis to detect mitotic cells

• Immunohistochemistry (IHC) for visualizing M-phase cells in tissue sections

• Western blotting (WB) to detect H3 pSer28 levels

• Immunofluorescence (IF) for cellular localization studies

• Chromatin immunoprecipitation (ChIP) to identify genomic regions associated with H3 pSer28

• ELISA for quantitative detection

These applications enable researchers to investigate the dynamics of histone H3 phosphorylation during various cellular processes, particularly during cell division and transcriptional regulation.

What are the optimal fixation protocols for preserving H3 pSer28 antigenicity in tissue samples?

Preserving H3 pSer28 antigenicity requires careful attention to fixation protocols. Research indicates that H3 pSer28 antigenicity is highly labile and can be rapidly lost during sample processing. Key methodological considerations include:

• Immediate fixation is crucial - a 30% reduction in HTA28 staining index was observed after soaking tissues in PBS for only 2 hours before fixation

• 10% neutral buffered formalin is recommended as the fixation agent

• Optimal fixation time is 24 hours at room temperature; extended fixation periods (48-168 hours) result in progressive loss of antigenicity

• Avoid repeat freeze-thaw cycles when storing antibody solutions

• For flow cytometry applications, use Protocol A (two-step protocol for cytoplasmic proteins) or Protocol C (two-step protocol with fixation/methanol) rather than Protocol B

These findings emphasize that surgeons and pathologists should immediately and precisely fix samples if clinical specimens are to be examined for H3 pSer28 .

How can I optimize western blotting protocols for HIST1H3A (Ab-28) antibody?

For optimal western blotting results with HIST1H3A (Ab-28) antibody:

To verify specificity, consider using:

• Dephosphorylated samples as negative controls

• Comparing with other H3 modification-specific antibodies to confirm band identity

What factors affect the reproducibility of flow cytometry results with HIST1H3A (Ab-28) antibody?

When using HIST1H3A (Ab-28) antibody for flow cytometry:

• Pre-titrate the antibody; optimal concentration is ≤0.5 μg per test (where a test refers to the amount needed to stain a cell sample in 100 μL)

• Cell number should be empirically determined but typically ranges from 10^5 to 10^8 cells/test

• For optimal detection, use Protocol A (two-step protocol for cytoplasmic proteins) or Protocol C (two-step protocol with fixation/methanol)

• Include both positive controls (e.g., nocodazole-treated cells with high mitotic index) and negative controls

• Use freshly prepared samples, as phosphorylation status can change during storage

• Ensure consistency in permeabilization conditions, as insufficient permeabilization can limit nuclear antigen access

How can HIST1H3A (Ab-28) antibody be used to study the relationship between histone phosphorylation and chromatin states?

Studies using H3 pSer28 antibodies have revealed important insights into chromatin regulation:

Research has shown that H3 phosphorylation at serine 28 preferentially partitions with chromatin enriched in transcriptionally active/competent regions. When chromatin was fractionated, H3S28p preferentially partitioned into fractions enriched in active and competent DNA sequences (F1 and F2), while H3S10p was equally distributed between all fractions . This suggests distinct roles for these two phosphorylation sites despite their proximity in the histone tail.

To investigate these relationships:

• Perform chromatin fractionation to separate active vs. inactive chromatin regions

• Use HIST1H3A (Ab-28) antibody in combination with antibodies against other histone marks in ChIP-seq experiments

• Correlate H3 pSer28 distribution with gene expression data to identify regulatory relationships

• Compare patterns of H3 pSer28 with other histone marks (H3K4me3, H3K27ac) associated with active chromatin

This approach enables researchers to map the genome-wide distribution of H3 pSer28 and correlate it with transcriptional activity and other chromatin features.

How reliable is HIST1H3A (Ab-28) antibody for quantifying mitotic cells compared to traditional methods?

The HTA28 monoclonal antibody has been extensively validated for detecting mitotic cells:

• In a study using regenerating rat liver after partial hepatectomy, the HTA28 staining index (SI) closely paralleled the mitotic index (MI) determined by morphological assessment, but was consistently higher at all time points

• HTA28 immunoreactivity was maintained throughout all stages of M-phase (prophase through telophase), making it more comprehensive than morphological assessment alone

• The spatial distribution of HTA28-positive cells corresponded with those identified by other proliferative cell markers

For optimal quantification:

• Include both positive controls (tissue with known high mitotic activity) and negative controls

• Consider dual staining with other cell cycle markers to confirm specificity

• Compare results with established methods (e.g., phospho-histone H3 Ser10, Ki-67) for validation

• Be aware that telophase cells may stain less intensely than earlier mitotic phases

What is the relationship between H3 pSer28 and H3 variant incorporation in chromatin remodeling?

Recent research has revealed complex relationships between histone phosphorylation and histone variant dynamics:

Studies show that histone variants H3.1 and H3.3 are differentially regulated and can be subject to distinct post-translational modifications. H3.1 serves as a chromatin redox sensor that is engaged by mitochondrial H2O2 signaling . The replacement of H3.1 by H3.3 has been correlated with increased chromatin accessibility and gene expression, particularly for EMT genes .

To investigate these relationships:

• Use HIST1H3A (Ab-28) antibody in combination with antibodies specific for H3.1 or H3.3 variants

• Perform ChIP-seq or CUT&RUN to map genome-wide distribution of pSer28 on specific H3 variants

• Correlate phosphorylation patterns with histone variant dynamics during processes like cell division or differentiation

• Use sequential ChIP (re-ChIP) to determine if pSer28 co-occurs with other modifications on the same histone molecule

This approach can provide insights into how phosphorylation at serine 28 might differentially affect the function of distinct histone H3 variants.

How can I verify the specificity of HIST1H3A (Ab-28) antibody in my experimental system?

To confirm antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with a synthetic phosphopeptide containing pSer28 (e.g., CKKAARKpSAPATGGV) before applying to your sample; this should eliminate specific signal

• Comparison with non-phosphorylated controls: Include samples treated with phosphatase or phosphorylation inhibitors

• Timing experiments: Since H3 pSer28 is cell cycle-dependent, synchronize cells and test at different cycle stages; the signal should increase during mitosis

• Cross-reactivity testing: Test against closely related modifications like H3 pSer10 to ensure specificity

• Knockout/knockdown validation: Where possible, use cells lacking the target or with reduced expression

It's worth noting that HTA28 specifically recognizes H3 phosphorylated at serine 28 but not serine 10, providing a useful tool for distinguishing between these modifications .

What are common pitfalls when using HIST1H3A (Ab-28) antibody and how can they be avoided?

Common challenges and their solutions include:

Additionally, always validate new lots of antibody against a known standard, and consider using recombinant antibodies when available for greater consistency.

How do I interpret conflicting results between HIST1H3A (Ab-28) antibody and other histone modification markers?

When facing discrepancies between H3 pSer28 and other histone modification patterns:

• Consider biological timing: Different modifications occur at distinct cell cycle phases or in response to different stimuli. H3S28 phosphorylation is mediated by MSK1 following activation of the MAP kinase signaling pathway , while other modifications may follow different pathways.

• Examine cross-talk between modifications: Modifications on neighboring residues can affect antibody recognition. For example, acetylation at K27 might impact recognition of pS28.

• Validate with multiple antibodies: Use antibodies from different sources or that recognize different epitopes containing the same modification.

• Consider technical factors: Different antibodies may require distinct fixation and antigen retrieval protocols for optimal performance.

• Employ complementary approaches: Combine antibody-based detection with mass spectrometry to resolve discrepancies.

Research has shown that H3 pSer28 and H3 pSer10 can have distinct distributions and functions despite their proximity in the histone tail , so apparent conflicts might reflect genuine biological differences rather than technical artifacts.

How can HIST1H3A (Ab-28) antibody be used to study the role of H3 pSer28 in disease processes?

Emerging applications of H3 pSer28 antibodies in disease research include:

• Cancer research: H3 pSer28 has been linked to chromosome condensation during mitosis, cell transformation, and regulation of RNA polymerase III transcription machinery . Aberrant phosphorylation patterns may contribute to genomic instability in cancer.

• Cell senescence studies: Histone modifications change dramatically during senescence. Research has identified distinct histone H3 tail proteolytic processing in models of both oncogene-induced and replicative senescence . HIST1H3A (Ab-28) antibody can help track changes in phosphorylation patterns during senescence induction.

• Neural development: Recent research has discovered that histone H3 monoaminylation dynamics regulate neural transcriptional programs, with H3Q5 identified as a primary site of modification . Studying the interplay between these modifications and phosphorylation at serine 28 could provide insights into neuronal differentiation and function.

• Therapeutic response monitoring: Changes in histone phosphorylation patterns can serve as biomarkers for response to treatment, particularly for drugs targeting kinase pathways that affect histone phosphorylation.

What are the considerations for multiplexing HIST1H3A (Ab-28) antibody with other antibodies in imaging or flow cytometry?

For successful multiplexing experiments:

• Antibody compatibility:

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • For same-species antibodies, use directly conjugated formats or sequential staining protocols

  • Consider using Zenon labeling technology for antibodies from the same species

• Fluorophore selection:

  • When using Alexa Fluor 488-conjugated HIST1H3A (Ab-28) antibody, select complementary fluorophores with minimal spectral overlap

  • For the Alexa Fluor 488 conjugate: Excitation: 488 nm; Emission: 519 nm

  • Pair with far-red fluorophores for maximum separation

• Staining protocol optimization:

  • Test antibodies individually before combining

  • Optimize concentration of each antibody separately

  • Consider sequential staining if antibodies require different fixation/permeabilization conditions

• Controls for multiplexing:

  • Include single-stained controls for compensation

  • Use fluorescence-minus-one (FMO) controls to set proper gates

  • Include isotype controls for each antibody

How does the binding of HIST1H3A (Ab-28) antibody affect the interpretation of chromatin dynamics studies?

When using HIST1H3A (Ab-28) antibody in chromatin studies, consider these advanced interpretational factors:

• Antibody accessibility issues: The compact nature of chromatin might limit antibody access to the H3 pSer28 epitope in certain chromatin states. This could lead to underestimation of modification levels in highly condensed regions.

• Epitope masking by protein complexes: Protein complexes that recognize H3 pSer28 might compete with antibody binding, potentially resulting in false negatives in regions where the modification is functionally engaged.

• Temporal dynamics considerations: H3 pSer28 is a dynamic modification whose levels change rapidly during cell cycle progression. Time-course studies with synchronized cells are essential for accurate interpretation.

• Single-cell vs. population heterogeneity: Flow cytometry or imaging approaches with HIST1H3A (Ab-28) antibody can reveal cell-to-cell variability that might be masked in bulk chromatin studies like ChIP-seq.

• Relationship to chromatin accessibility: Studies have shown that H3 phosphorylation at serine 28 preferentially partitions with chromatin enriched in transcriptionally active/competent regions . This association should be considered when interpreting genomic distribution patterns.