Phospho-Histone H3(Ser28) Monoclonal Antibody
Description
Target and Biological Significance
Phospho-Histone H3(Ser28) monoclonal antibodies specifically recognize histone H3 when phosphorylated at serine 28, a 15 kDa protein critical for chromatin structure and nucleosome assembly . Ser28 phosphorylation is mediated by MSK1 following activation by the MAP kinase pathway in response to stress signals (e.g., UV, EGF) or oncoproteins (e.g., c-Myc) . Unlike serine 10 phosphorylation (a well-established mitotic marker), Ser28 phosphorylation occurs exclusively during prophase and is absent in interphase cells . It correlates with chromosome condensation, RNA polymerase III regulation, and neoplastic transformation .
Mitotic Index Quantification
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Flow cytometry: Used to assess mitotic arrest in nocodazole-treated HeLa cells, with optimal staining at 0.25–0.5 µg/test .
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Immunofluorescence: Localizes to condensed chromosomes in prophase cells .
Cancer Research
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Comparative studies: Ser28 phosphorylation is less prevalent than Ser10 in untreated (0.5% vs. 4.75%) and nocodazole-treated HeLa cells (30.1% vs. 51.2%) .
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Clinical relevance: Lower Ser28 levels in cancers suggest it is a secondary mitotic marker compared to Ser10 .
Drug Response Studies
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ELISA kits (e.g., Active Motif 53100) quantify Ser28 phosphorylation in acid-extracted histones, detecting as little as 156 ng of extract .
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Paclitaxel-treated cells show elevated Ser28 phosphorylation due to mitotic arrest .
Staining Optimization
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Fixation: Methanol or paraformaldehyde fixation preserves epitope integrity .
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Permeabilization: 0.1% Triton X-100 enhances antibody access .
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Controls: Include nocodazole-treated (positive) and thymidine-synchronized (negative) cells .
Limitations
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Lower sensitivity: Ser28 antibodies detect fewer mitotic cells than Ser10 antibodies, potentially due to delayed phosphorylation timing or epitope accessibility .
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Species restrictions: Some clones (e.g., Cell Signaling #9713) are validated only for human, mouse, and hamster .
Emerging Insights
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Data indicate the mechanism for epigenetic regulation in cancer by inducing E3 ubiquitin ligase NEDD4-dependent histone H3 ubiquitination. PMID: 28300060
- The identification of increased expression of H3K27me3 during a patient's clinical course can be helpful for determining whether the tumors are heterochronous. PMID: 29482987
- We report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. PMID: 28982940
- Data suggest that the Ki-67 antigen proliferative index has significant limitations, and phosphohistone H3 (PHH3) is an alternative proliferative marker. PMID: 29040195
- These results identify cytokine-induced histone 3 lysine 27 trimethylation as a mechanism that stabilizes gene silencing in macrophages. PMID: 27653678
- This data indicates that, in the early developing human brain, HIST1H3B constitutes the largest proportion of H3.1 transcripts among H3.1 isoforms. PMID: 27251074
- This series of 47 diffuse midline gliomas reveals that the histone H3-K27M mutation was mutually exclusive with IDH1-R132H mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss, and monosomy 10. Among these K27M+ diffuse midline gliomas. PMID: 26517431
- Data show that histone chaperone HIRA co-localizes with viral genomes, binds to incoming viral DNA, and deposits histone H3.3 onto these. PMID: 28981850
- These experiments showed that PHF13 binds specifically to DNA and to two types of histone H3 methyl tags (lysine 4-tri-methyl or lysine 4-di-methyl) where it functions as a transcriptional co-regulator. PMID: 27223324
- Hemi-methylated CpGs DNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. PMID: 27595565
- We describe, for the first time, the MR imaging features of pediatric diffuse midline gliomas with histone H3 K27M mutation. PMID: 28183840
- Approximately 30% of pediatric high-grade gliomas (pedHGG) including GBM and DIPG harbor a lysine 27 mutation (K27M) in histone 3.3 (H3.3) which is correlated with poor outcome and was shown to influence EZH2 function. PMID: 27135271
- H3F3A K27M mutation in adult cerebellar HGG is not rare. PMID: 28547652
- Data show that lysyl oxidase-like 2 (LOXL2) is a histone modifier enzyme that removes trimethylated lysine 4 (K4) in histone H3 (H3K4me3) through an amino-oxidase reaction. PMID: 27735137
- Histone H3 lysine 9 (H3K9) acetylation was most prevalent when the Dbf4 transcription level was highest whereas the H3K9me3 level was greatest during and just after replication. PMID: 27341472
- SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing. PMID: 27614073
- Data suggest that binding of the helical tail of histone 3 (H3) with PHD ('plant homeodomain') fingers of BAZ2A or BAZ2B (bromodomain adjacent to zinc finger domain 2A or 2B) requires molecular recognition of secondary structure motifs within the H3 tail and could represent an additional layer of regulation in epigenetic processes. PMID: 28341809
- The results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate the formation of the preinitiation complex. PMID: 27679476
- Histone H3 modifications caused by traffic-derived airborne particulate matter exposures in leukocytes. PMID: 27918982
- A key role of persistent histone H3 serine 10 or serine 28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response genes. PMID: 27996159
- hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in medulloblastoma. PMID: 27694758
- AS1eRNA-driven DNA looping and activating histone modifications promote the expression of DHRS4-AS1 to economically control the DHRS4 gene cluster. PMID: 26864944
- Data suggest that nuclear antigen Sp100C is a multifaceted histone H3 methylation and phosphorylation sensor. PMID: 27129259
- The authors propose that histone H3 threonine 118 phosphorylation via Aurora-A alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. PMID: 26878753
- Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its H3 histone recognition. PMID: 27045799
- Functional importance of H3K9me3 in hypoxia, apoptosis and repression of APAK. PMID: 25961932
- Taken together, the authors verified that histone H3 is a real substrate for GzmA in vivo in the Raji cells treated by staurosporin. PMID: 26032366
- We conclude that circulating H3 levels correlate with mortality in sepsis patients and inversely correlate with antithrombin levels and platelet counts. PMID: 26232351
- Data show that double mutations on the residues in the interface (L325A/D328A) decreases the histone H3 H3K4me2/3 demethylation activity of lysine (K)-specific demethylase 5B (KDM5B). PMID: 24952722
- Data indicate that minichromosome maintenance protein 2 (MCM2) binding is not required for incorporation of histone H3.1-H4 into chromatin but is important for stability of H3.1-H4. PMID: 26167883
- Data suggest that histone H3 lysine methylation (H3K4me3) serves a crucial mechanistic role in leukemia stem cell (LSC) maintenance. PMID: 26190263
- PIP5K1A modulates ribosomal RNA gene silencing through its interaction with histone H3 lysine 9 trimethylation and heterochromatin protein HP1-alpha. PMID: 26157143
- Data indicate that the lower-resolution mass spectrometry instruments can be utilized for histone post-translational modifications (PTMs) analysis. PMID: 25325711
- Data indicate that inhibition of lysine-specific demethylase 1 activity prevented IL-1beta-induced histone H3 lysine 9 (H3K9) demethylation at microsomal prostaglandin E synthase 1 (mPGES-1) promoter. PMID: 24886859
- The authors report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. PMID: 22473132
Q&A
How Do I Validate the Specificity of the Phospho-Histone H3(Ser28) Antibody for My Experimental System?
Validating antibody specificity requires rigorous controls and comparative analysis:
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Positive Controls: Use nocodazole-treated cells (e.g., HeLa) to synchronize cells in mitosis, where H3S28 phosphorylation peaks . Confirm signal localization to mitotic chromatin.
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Negative Controls: Include PTX-treated extracts (Active Motif ELISA) to exclude non-specific binding .
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Cross-Reactivity Testing: Confirm no signal in samples where Ser28 phosphorylation is inhibited (e.g., MLTK-alpha knockdown cells) .
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Western Blot vs. IHC/Flow Cytometry:
What Distinguishes Phospho-Histone H3(Ser28) from Phospho-Histone H3(Ser10) in Experimental Applications?
Key Differences:
Advanced Consideration: Co-detection of H3S28p with H3K4me3 or H3.3 variants can distinguish transcriptional activation from mitotic events .
How Should I Optimize Intracellular Staining Protocols for Flow Cytometry?
Protocol Recommendations:
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Fixation: Use 4% paraformaldehyde (10 min) followed by methanol permeabilization (30 min at -20°C) .
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Antibody Titration: Start with 5 µL/test (0.25 µg) for HeLa cells; adjust for cell density (10⁵–10⁸ cells/test) .
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Fluorophore Compatibility: Use eFluor® 660 (excitation 633–647 nm) to avoid spectral overlap with other markers .
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Controls: Include isotype-matched antibodies and untreated samples to assess background noise.
Troubleshooting:
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Low Signal: Check fixation time (excessive fixation may mask epitopes).
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High Background: Use blocking buffers (e.g., 2% BSA) and validate with H3S28p-negative cell lines .
What Controls Should Be Included in Phospho-Histone H3(Ser28) ELISA to Ensure Reliable Quantification?
Critical Controls:
Data Analysis:
How Does H3S28 Phosphorylation Correlate with Chromatin Dynamics in Transcriptionally Active Regions?
Mechanistic Insights:
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Active Promoters: H3S28p is enriched in transcriptionally competent chromatin (S150 fraction) alongside H3K4me3 and H3.3 variants .
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Nucleosome Destabilization: H3.3 phosphorylated at Ser28 forms labile nucleosomes, facilitating dynamic chromatin remodeling .
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Transcriptional Activation: Linked to RNA Pol III recruitment, distinct from mitotic H3S10p .
Experimental Approach:
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Co-stain with H3.3 antibodies to assess variant-specific phosphorylation.
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Use chromatin fractionation (e.g., S150 vs. S200) to isolate active vs. repressed regions .
What Signaling Pathways Regulate H3S28 Phosphorylation, and How Can They Be Manipulated Experimentally?
Key Pathways and Inhibitors:
| Stimulus | Kinase Involved | Inhibitor Test | Source |
|---|---|---|---|
| UVB/EGF | MLTK-alpha | MLTK-alpha siRNA | |
| c-Myc/c-Jun | MSK1 | H89 (PKA inhibitor) | |
| Tumor Promoters | MAPK cascade | PD98059 (MEK inhibitor) |
Advanced Strategy:
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Use UVB (10 mJ/cm²) or EGF (100 ng/mL) to induce H3S28p in JB6 Cl41 cells; validate with MLTK-alpha knockdown .
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Confirm pathway specificity by testing inhibitors (SB202190 for p38; ineffective for MLTK-alpha) .
How Do I Resolve Discrepancies in H3S28 Phosphorylation Levels Across Studies?
Common Sources of Variation:
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Antibody Specificity:
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Cell Cycle Synchronization:
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Sample Preparation:
Data Reconciliation:
What Implications Does the Use of H3.3 Variants Have for Ser28 Phosphorylation Detection?
H3.3-Specific Findings:
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Phosphorylation Preference: H3.3 variants are preferentially phosphorylated at Ser28 in active chromatin .
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Nucleosome Stability: H3.3-S28p nucleosomes are labile, aiding transcriptional activation .
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Experimental Design:
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Co-detect H3.3 and H3S28p via sequential IHC or ChIP-seq.
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Use H3.3 knockout cells to assess variant-specific contributions.
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How Can I Distinguish the Role of H3S28 Phosphorylation in Mitosis vs. Transcriptional Regulation?
Experimental Separation:
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Mitotic Context:
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Transcriptional Context:
Data Interpretation:
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H3S28p in mitosis: Correlates with chromosome condensation .
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H3S28p in transcription: Enriched at gene promoters with H3.3 and H3K4me3 .
