Phospho-Histone H3 (Ser10), Phospho-Histone H3 (Thr3) & Total Histone H3 Cellular Assay Kits

HTRF® cell-based phospho and total-Histone H3 assays for anti-cancer research

The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination. These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression. Phosphorylation of Ser10 or Thr3 in the tails of Histone H3 is involved in gene expression and during mitosis.

Phosphorylation at Ser10 is tightly correlated with chromosome condensation during both mitosis and meiosis.

Phosphorylation at Threonine 3 catalyzed by the kinase haspin and highly conserved among many species leads to recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores, to regulate mitosis.

Cisbio's cell-based homogeneous phospho-Histone H3 assay kits are designed for the cell-based quantitative detection of phosphorylation on Histone H3. The HTRF® total Histone H3 assay kit is designed for the quantitative detection of total Histone H3, phosphorylated and unphosphorylated, to normalize the phosphorylation status of Histone H3.

The buffers of both HTRF® phospho- and total Histone H3 assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

   Check our lysis buffer compatibility      Check our species compatibility

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total Histone H3 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-Histone H3 antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with Histone H3, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal.

The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-Histone H3 can be quantitatively detected using the HTRF phospho-Histone H3 (Thr3), phospho-Histone H3 (Ser10) and total Histone H3 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well small volume assay plate where the HTRF reagents are added. This also enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

The protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol means enhanced speed and simplicity, enabling all throughputs and fast results while maintaining high quality and sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

Histone3 is one of the 5 major canonical Histone proteins to compact DNA into chromatin. The main globular histone domains remain in theoctameric nucleosome core, from which the long N-terminal Histone tails extend. These Histone protein tails are highly post-translationallymodified, and are responsible for the constitution of either highly condensed heterochromatin or less condensed euchromatin. The different types of coordinated modifications, like acetylation, methylation and phosphorylation, compose the histone code, read by the remodeling machinery. Thus Histone modifications play a major role in the dynamic and long term regulation of genes, making the surrounding DNA more or less accessible to the transcriptional machinery. Phosphorylation of Ser10 or Thr3 in the tails of Histone H3 is involved in gene expression and during mitosis.

Phospho-Histone H3-Ser10 in the cell cycle

The overall level of histone phosphorylation fluctuates very markedly during the cell cycle and is characterized by the existence of peaks in phosphorylation of Histone H3 on Serine10 in mitosis. The Aurora kinases A, B & C are key regulators of mitosis, and Aurora kinase B is directly responsible for the mitotic Histone H3 Ser10 phosphorylation. Aurora kinase A, in complex with BORA, is responsible for the initial activation of PLK1 in G2 phase, leading to the activation of cyclin-dependent kinase 1 mitotic entry at the G2/M transition checkpoint. Aurora kinase A is frequently amplified in human cancers, and Aurora kinase A overexpression in human tumours correlates with a poor prognosis and genomic instability. Moreover, high expression levels of Aurora kinase B are associated with a poor prognosis in glioblastoma, ovarian carcinoma and hepatocellular carcinoma. Another kinase, PAK-1 (p21-activated kinase-1), has also been implicated in Histone H3 Ser10 phosphorylation.

Phospho-Histone H3-Thr3 in cell signaling

There are four characterized residues within the H3 N-terminal tail that are phosphorylated during mitosis: Thr3, Ser10, Thr11, and Ser28. There is a precise spatio-temporal correlation between Thr3 phosphorylation and stages of mitosis: the phosphorylation starts at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. Phosphorylation at Thr3 is highly conserved among many species and is catalyzed by the kinase Haspin. Thr3 phosphorylation then provides a chromatin binding site for the recruitment of the chromosomal passenger complex (CPC) which consists of four proteins: Survivin which binds to H3, Aurora-B, INCEMP and Borealin. The Aurora kinase B then phosphorylates Haspin, which creates a signal amplification loop and contributes to CPC accumulation at centromeres to regulate mitosis and chromosome segregation.The dephosphorylation is catalyzed by the complex PP1γ/Repo-Ma.

Product Performances

1. HTRF® phospho-S10 assay compared to Western Blot

Exponentially growing Hela cells were cultivated in a T175cm2 flask, until 80% confluency. Hela cells were treated with Nocodazole for 16 hours. After removal of cell culture medium, 3 ml of supplemented lysis buffer were added and incubated for 30 minutes. Soluble supernatants were collected after a10-minute centrifugation. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.

Using HTRF® phospho-Histone H3 (Ser10), only 256 cells are sufficient for minimal signal detection, while 6,400 cells are needed for a Western Blot signal. The HTRF® assay is 25-fold more sensitive than the Western Blot.

2. Phospho-Histone H3-S10 as a mitotic cell cycle biomarker for cell proliferation

Hela and A431 cells were seeded in a 96-well plate and treated for 16 hours with Nocodazole, an antimitotic agent that depolymerizes microtubules and blocks the cells in mitosis, when Histone H3 S10 phosphorylation is maximal. After cell culture media removal, 50 µl, 100 µl or 200 µl of lysis buffer were added for a 30-minute incubation. 16 µl of each lysate were transferred to a 384sv white plate for HTRF analysis. Increasing the lysis volume up to 200µl improved the linearity of the HTRF detection signal.

3. Pharmacological response on phospho-Histone H3-S10 as read-out for inhibition of Aurora Kinase B activity

Hela, Jurkat and NIH3T3 cells were seeded in a 96-well plate and treated with Nocodazole, an antimitotic agent that depolymerize microtubules and blocks the cells in mitosis, when Histone H3 S10 phosphorylation is maximal. The cells were co-treated for 16 hours with the Aurora Kinase B inhibitors, either with increasing concentrations of Danusertib (PHA-739358) on Hela and Jurkat cells, or Alisertib (MLN8237) on NIH3T3 cells. After cell culture media removal, 200 µl of lysis buffer were added for a 30-minute incubation. 16 µl of each lysate were transferred to a 384sv white plate for HTRF analysis.

 

 

4. Total-Histone H3 as normalization control for phospho-Histone H3 read-out

NIH3T3 cells (12.5KC per well) were seeded in a 96-well plate and treated with Nocodazole, an antimitotic agent that depolymerizes microtubules and blocks the cells in mitosis, to stimulate Histone H3 S10 phosphorylation. Cells were (experiment a) or weren’t (experiment b) co-treated for 16 hours with an inhibitor Danusertib. After cell culture medium removal, 200 µL of supplemented lysis buffer were added for a 30-minute incubation. 10 µL of lysates each were transferred to a 384sv white plate for HTRF analysis in parallel with the total and the phospho-Ser10 assays

a. Stimulation of Histone H3 phosphorylation by Nocodazole

b. Pharmacological response of Danusertib inhibitor on Histone H3 phosphorylation after stimulation by nocodazole

The cells were co-treated for 16 hours with the Aurora Kinase B inhibitor Danusertib (PHA-739358).

5. HTRF assay compared to Western Blot using phospho H3 (Thr3) cellular assay

Human cervical cancer HeLa cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2 until reaching 80% confluency. After treatment for 30 min at 37°C with 200 nM Calyculin A, cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. The soluble fractions was were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, then 16 µL of neat or diluted fractions were dispensed and analyzed side-by-side by Western Blot and by HTRF.

The HTRF phospho H3 (Thr3) assay is at least 16-fold more sensitive than the Western Blot. Using HTRF, 21,000 cells are sufficient for minimal signal detection while 32,000 cells are needed for a Western Blot signal.

6. Validation of the HTRF phospho H3 (Thr3) cellular assay on mouse and human cell lines

50,000 human cervical cancer HeLa cells or mouse fibroblast NIH-3T3 cells were plated in 96-well plates and incubated for 24h at 37 °C - 5% CO2. Cells were stimulated at 37°C for 30 min with increasing concentration of Calyculin-A. Medium was then removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-Histone H3 (Thr3) detection reagents were added. The HTRF signal was recorded after a 2h incubation time at room temperature.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Histone H3 phospho-T3 kit - 500 tests63ADK061PEG
-
Histone H3 phospho-T3 kit - 10,000 tests63ADK061PEH
-
Histone H3 phospho-T3 kit - 50,000 tests63ADK061PEY
-
Histone H3 phospho-S10 kit - 500 tests64HH3PEG
-
Histone H3 phospho-S10 kit -10,000 tests64HH3PEH
-
Histone H3 phospho-S10 kit - 50,000 tests64HH3PEY
-
Histone H3 total kit - 500 tests64NH3PEG
Histone H3 total kit - 10,000 tests64NH3PEH
Histone H3 total kit - 50,000 tests64NH3PEY
-

Companion products

DescriptionCat. noProduct insertMSDS
Histone H3 phospho-T3 kit control lysate63ADK061TDA
-
Histone H3 phospho-S10 kit control lysate64HH3TDA
-
-
Histone H3 total kit control lysate64NH3TDA
-
-

Documents