HIST1H3A (Ab-28) Antibody

What is HIST1H3A (Ab-28) Antibody and what epitope does it recognize?

HIST1H3A (Ab-28) Antibody is a specialized antibody that recognizes histone H3 phosphorylated at serine 28 (pSer28). This antibody specifically binds to the phosphorylated form of serine 28 on histone H3 but does not cross-react with the phosphorylated serine 10 site or non-phosphorylated histone H3 . The antibody is typically generated using synthetic phosphorylated peptides as antigens to ensure specificity to the phospho-serine 28 epitope . The immunogen used is generally a synthesized peptide derived from Human Histone H3 around the phosphorylation site of S28 . This high specificity makes it an excellent marker for detecting cells undergoing mitosis, as histone H3 phosphorylation at serine 28 is closely associated with chromosomal condensation during the M-phase of the cell cycle.

What are the primary applications of HIST1H3A (Ab-28) Antibody?

The primary applications of HIST1H3A (Ab-28) Antibody include:

• Western Blotting (WB): For detecting phosphorylated histone H3 in protein samples

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

• Immunofluorescence (IF): For identifying mitotic cells in cultured cells or tissue sections

• ELISA: For quantitative assessment of phosphorylated histone H3

• Cell cycle analysis: Specifically for identifying and quantifying cells in M-phase

This antibody is particularly valuable in proliferation studies, as it allows researchers to distinguish M-phase cells from cells in other stages of the cell cycle with high specificity. Unlike general proliferation markers that identify cells in multiple phases, HIST1H3A (Ab-28) Antibody specifically marks cells undergoing mitosis, providing more precise analysis of cell division events .

How does phosphorylation at serine 28 correlate with cell cycle progression?

Phosphorylation of histone H3 at serine 28 is a highly specific marker for cells in mitosis. The phosphorylation begins during prophase, coinciding with the initiation of chromosomal condensation, and is maintained throughout all stages of M-phase until telophase . This post-translational modification plays a critical role in chromatin remodeling during mitosis.

The temporal pattern shows that:

• Phosphorylation begins in prophase

• Reaches maximum intensity during metaphase and early anaphase

• Gradually decreases during telophase

• Is completely absent in interphase cells

Studies in regenerating rat liver after partial hepatectomy have demonstrated that the HIST1H3A (Ab-28) staining index closely follows the mitotic index but with higher absolute values, confirming its utility as a sensitive M-phase marker . This correlation makes the antibody particularly useful for studying cell proliferation kinetics in developmental biology, cancer research, and regenerative medicine.

What are the optimal sample preparation conditions for HIST1H3A (Ab-28) Antibody staining?

Optimal sample preparation is crucial for successful HIST1H3A (Ab-28) Antibody staining, as the phospho-epitope is sensitive to both delayed fixation and prolonged fixation. Based on experimental evidence, the following guidelines should be followed:

• Immediate fixation: Tissues should be fixed immediately after collection to preserve phosphorylation at serine 28. Studies have shown that soaking tissues in PBS for just 2 hours before fixation can result in a 30% reduction in staining index .

• Fixation duration: Optimal fixation is achieved with 10% neutral buffered formalin for 24 hours at room temperature. Extended fixation times progressively reduce antigenicity .

• Sectioning: Thin sections (2-3 μm) provide optimal results for visualization of nuclear details .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended to enhance antibody binding.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody to reduce background staining.

It's important to note that approximately 3.4% of mitotic cells can lose their reactivity even with optimal 24-hour fixation, highlighting the sensitivity of this phospho-epitope . For clinical specimens, immediate and precise fixation is strongly recommended to preserve antigenicity.

How can I optimize detection protocols for HIST1H3A (Ab-28) Antibody in different experimental systems?

Optimizing detection protocols for HIST1H3A (Ab-28) Antibody requires consideration of several factors depending on the experimental system:

For Immunohistochemistry:

• Antibody dilution: Start with 1:100-1:500 and optimize based on signal-to-noise ratio

• Incubation time: Typically 1-2 hours at room temperature or overnight at 4°C

• Detection system: 3,3'-diaminobenzidine (DAB) provides good contrast for nuclear staining

• Counterstain: Light hematoxylin counterstaining helps visualize negative nuclei without obscuring positive staining

For Immunofluorescence:

• Use higher antibody concentrations for weaker signals in telophase cells

• Consider tyramide signal amplification for detecting low levels of the phospho-epitope

• Combine with DAPI to visualize all nuclei

For Western Blotting:

• Use freshly prepared lysates to preserve phosphorylation

• Include phosphatase inhibitors in all buffers

• Positive controls: Include mitotic cell extracts (e.g., nocodazole-treated cells)

• Negative controls: Include interphase cell extracts and phosphatase-treated samples

The reactivity of HIST1H3A (Ab-28) Antibody can be enhanced by increasing antibody concentration or extending development time with chromogens, which can be particularly useful for detecting cells in telophase that typically show weaker staining .

How does HIST1H3A (Ab-28) Antibody compare with other mitotic markers in experimental systems?

HIST1H3A (Ab-28) Antibody offers distinct advantages compared to other mitotic and proliferation markers:

Studies comparing HIST1H3A (Ab-28) staining with traditional mitotic indices have shown that the antibody consistently detects approximately twice as many M-phase cells compared to conventional H&E staining . This is because HIST1H3A (Ab-28) detects cells in all mitotic stages, while traditional mitotic counting often focuses on metaphase and anaphase figures only.

Additionally, HIST1H3A (Ab-28) Antibody shows a temporal pattern similar to the BrdU labeling index but with a time lag, consistent with cell cycle progression from S-phase (detected by BrdU) to M-phase . This relationship makes it valuable for comprehensive cell cycle studies when used in combination with other phase-specific markers.

What are the dynamics of histone H3 serine 28 phosphorylation throughout mitosis?

The dynamics of histone H3 serine 28 phosphorylation show a specific pattern throughout mitosis that correlates with chromosomal condensation:

• Initiation: Phosphorylation at serine 28 begins during prophase, coinciding with the initial stages of chromosomal condensation .

• Maintenance: The phosphorylation is maintained at high levels throughout prophase, metaphase, and early anaphase, with maximum intensity during metaphase when chromosomes are fully condensed .

• Decline: Phosphorylation begins to decrease during anaphase and weakens significantly during telophase .

• Absence: Complete dephosphorylation occurs by the end of telophase, with interphase cells showing no reactivity .

This dynamic pattern reflects the role of histone H3 phosphorylation in chromosomal condensation and segregation during mitosis, and understanding these dynamics is essential for accurate interpretation of experimental results.

How can HIST1H3A (Ab-28) Antibody be used in combination with other markers for comprehensive cell cycle analysis?

Combining HIST1H3A (Ab-28) Antibody with other cell cycle markers allows for comprehensive analysis of proliferation dynamics:

Recommended Marker Combinations:

• For complete cell cycle analysis:

  • HIST1H3A (Ab-28) for M-phase

  • BrdU or EdU for S-phase

  • Cyclin D1 for G1-phase

  • Cyclin B1 for G2-phase

• For mitotic subphase discrimination:

  • HIST1H3A (Ab-28) for all mitotic cells

  • Aurora B for chromosomal passenger complex visualization

  • α-tubulin for spindle formation

• For proliferation vs. apoptosis studies:

  • HIST1H3A (Ab-28) for mitotic cells

  • Ki-67 for all proliferating cells

  • Cleaved caspase-3 for apoptotic cells

Methodological Approach:

• Use serial sections for IHC with different antibodies

• For co-immunofluorescence, select antibodies from different host species

• Employ spectral unmixing for multiple fluorophores

• Consider sequential staining protocols for antibodies from the same species

In studies of regenerating rat liver, the temporal patterns showed that BrdU labeling (S-phase) precedes HIST1H3A (Ab-28) positivity (M-phase) by approximately 6-8 hours, consistent with normal cell cycle progression . This time relationship can be used to estimate cell cycle duration in various tissues and experimental conditions.

What are common issues with HIST1H3A (Ab-28) Antibody staining and how can they be resolved?

Several common issues can arise with HIST1H3A (Ab-28) Antibody staining, each requiring specific troubleshooting approaches:

Experimental evidence has shown that antigenicity for HIST1H3A (Ab-28) can be significantly compromised if tissue fixation is delayed, with a 30% reduction in staining after just 2 hours delay before fixation . Additionally, extended fixation times progressively reduce immunoreactivity, emphasizing the importance of standardized sample preparation protocols .

For clinical specimens where immediate fixation may be challenging, consider using alternative fixatives or rapid fixation protocols specifically optimized for phospho-epitope preservation.

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

Validating the specificity of HIST1H3A (Ab-28) Antibody is crucial for reliable experimental results. Consider the following validation approaches:

• Peptide competition assays:

  • Pre-incubate the antibody with phosphorylated peptide (PH28: CKKAARKpSAPATGGV)

  • Pre-incubate with non-phosphorylated peptide (H28: CKKAARKSAPATGGV) as control

  • Specific binding should be blocked only by the phosphorylated peptide

• Phosphatase treatment controls:

  • Treat one set of samples with lambda phosphatase before antibody application

  • Compare to untreated samples

  • Signal should be eliminated in phosphatase-treated samples

• Correlation with mitotic morphology:

  • Compare antibody staining with morphological identification of mitotic cells

  • HIST1H3A (Ab-28) positive cells should correspond to cells with mitotic morphology in H&E staining

  • Staining index should be approximately twice the conventional mitotic index

• Knockout or knockdown validation:

  • Use genetic approaches (if available) to knockout or knockdown histone H3

  • Alternatively, use CRISPR to mutate the serine 28 residue to alanine

  • Specific staining should be absent in these systems

• Cell cycle synchronization:

  • Compare staining in synchronized populations (G1/S vs. M-phase)

  • Positive staining should be restricted to M-phase cells

  • No staining should be observed in interphase cells

It's important to note that HTA28 antibody (which recognizes H3 phosphorylated at serine 28) has been confirmed to react with the synthetic peptide PH28 but not with H28, confirming its phospho-specificity . Similar validation should be performed for any HIST1H3A (Ab-28) Antibody used in research to ensure reliable results.

How can HIST1H3A (Ab-28) Antibody be applied in cancer research?

HIST1H3A (Ab-28) Antibody offers valuable applications in cancer research, particularly for assessing proliferation dynamics and treatment responses:

• Tumor proliferation assessment:

  • Quantify mitotic index with higher sensitivity than conventional H&E

  • HIST1H3A (Ab-28) detects approximately twice as many M-phase cells as conventional mitotic counting

  • Provides more accurate proliferation assessment in tumor grading systems

• Treatment response monitoring:

  • Evaluate anti-mitotic drug efficacy by quantifying changes in M-phase cells

  • Monitor temporal dynamics of cell cycle arrest and recovery

  • Distinguish between cytostatic and cytotoxic effects

• Spatial heterogeneity analysis:

  • Map proliferation hotspots within tumors

  • Correlate with other markers (hypoxia, stem cell markers, etc.)

  • Identify regions of aggressive growth for targeted therapies

• Cell cycle checkpoint studies:

  • Investigate mitotic checkpoint alterations in cancer

  • Combine with checkpoint proteins (Mad2, BubR1) to assess mitotic regulation

• Chromatin dynamics research:

  • Study altered histone modification patterns in cancer

  • Investigate epigenetic dysregulation in chromosomal instability

The application of HIST1H3A (Ab-28) Antibody in cancer research allows for precise quantification of M-phase cells, providing valuable information about tumor proliferation rates that correlate with aggressiveness and prognosis. The spatial distribution of positive cells can be analyzed to assess intratumoral heterogeneity, which is increasingly recognized as a critical factor in treatment resistance and disease progression.

What are the considerations for using HIST1H3A (Ab-28) Antibody in developmental biology studies?

In developmental biology, HIST1H3A (Ab-28) Antibody can provide valuable insights into proliferation patterns during embryogenesis and tissue differentiation. Special considerations include:

• Tissue-specific fixation protocols:

  • Embryonic tissues may require shorter fixation times

  • Optimize fixation based on developmental stage and tissue type

  • Consider specialized fixatives for delicate embryonic structures

• Developmental stage variations:

  • Baseline mitotic rates vary significantly between developmental stages

  • Compare to stage-matched controls rather than adult tissues

  • Consider circadian rhythms in proliferation for accurate comparisons

• Spatial proliferation mapping:

  • Use HIST1H3A (Ab-28) to map proliferative zones in developing tissues

  • Correlate with expression of developmental regulators

  • Track changes in proliferation patterns during morphogenesis

• Combinatorial approaches:

  • Combine with lineage markers to identify proliferating progenitor populations

  • Use with differentiation markers to study transition from proliferation to differentiation

  • Pair with apoptosis markers to assess tissue remodeling dynamics

• Quantification methods:

  • Develop standardized counting approaches for complex 3D structures

  • Consider volume-based rather than area-based quantification

  • Use digital image analysis for consistent assessment across developmental stages

In developmental studies, the high specificity of HIST1H3A (Ab-28) Antibody for M-phase cells makes it particularly valuable for precisely mapping proliferative zones and understanding the relationship between cell cycle dynamics and morphogenesis. The antibody can help identify regions of active cell division during critical developmental periods, providing insights into the mechanisms of tissue formation and growth regulation.