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Preparation And Storage
Recommended Assay Procedures
Recommended Assay Procedure
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and
culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either cold methanol or Triton™ X-100:
a. Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
Triton is a trademark of The Dow Chemical Company.
4. Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 µl of blocking buffer (3% FBS in 1× PBS) or BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well and incubating for 30 minutes at RT.
6. Remove the blocking buffer, dilute the antibody conjugate 1:10 in blocking buffer or Stain Buffer (FBS), and stain the cells by adding 50 µl of the diluted antibody conjugate to each well and incubating for 1 hour at RT.
7. Remove the diluted antibody conjugate, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
9. View and analyze the cells on an appropriate imaging instrument.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
Histones are highly basic proteins that complex with DNA to form chromatin. Histone H3 is a ~15-kDa protein that is phosphorylated at serine 28 (S28), S10, and/or threonine 11 during mammalian cell mitosis and meiosis. The phosphorylation sites are located in the N-terminal tail, a region that is outside of the chromatin fiber and is thus accessible for interactions with agents that may regulate chromatin or specific gene activities. The phosphorylation states of the two serine sites during the cell cycle are highly regulated by Aurora B kinase and a PP1 phosphatase: S10 is in the phosphorylated state from late G2 phase to anaphase, while S28 is phosphorylated from prophase to anaphase. Furthermore, phosphorylation of histone H3 S28 may be mediated by other kinases in response to external stimuli. Evidence suggests that histone phosphorylation is involved in the regulation of chromosome condensation, cell division, and gene transcription.
The HTA28 monoclonal antibody reacts with histone H3 phosphorylated at S28 in its N-terminal tail. It does not recognize the unphosphorylated protein.
Development References (9)
-
Carmena M, Earnshaw WC. The cellular geography of aurora kinases. Nat Rev Mol Cell Biol. 2003; 4:842-854. (Biology).
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Cheung P, Allis CD, Sassone-Corsi P. Signaling to chromatin through histone modifications. Cell. 2000; 103:263-271. (Biology).
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Choi HS, Choi BY, Cho Y-Y, Zhu F, Bode AM, Dong Z. Phosphorylation of Ser28 in histone H3 mediated by mixed lineage kinase-like mitogen-activated protein triple kinase α. J Biol Chem. 2005; 280(14):13545-13553. (Biology).
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Dyson MH, Thomson S, Inagaki M, et al. MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2. J Cell Sci. 2005; 118(10):2247-2259. (Clone-specific: Immunofluorescence).
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Furukawa K, Sugiyama S, Osouda S, et al. Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila. J Cell Sci. 2003; 116(18):3811-3823. (Clone-specific).
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Goto H, Tomono Y, Ajiro K, et al. Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. J Biol Chem. 1999; 274(36):25543-25549. (Immunogen: Immunofluorescence).
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Hirata A, Inada K, Tsukamoto T, et al. Characterization of a monoclonal antibody, HTA28, recognizing a histone H3 phosphorylation site as a useful marker of M-phase cells. J Histochem Cytochem. 2004; 52(11):1503-1509. (Clone-specific).
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Nowak SJ, Corces VG. Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet. 2004; 20(4):214-220. (Biology).
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Pascreau G, Arlot-Bonnemains Y, Prigent C. Phosphorylation of histone and histone-like proteins by aurora kinases during mitosis. Prog Cell Cycle Res. 2003; 5:369-374. (Biology).
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
