Product Pathways - Chromatin Regulation / Epigenetics
Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb #13576
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Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, F=Flow Cytometry, IF-IC=Immunofluorescence (Immunocytochemistry), ChIP=Chromatin IP,
Species predicted to react based on 100% sequence homology: Rat, Zebrafish, S. cerevisiae,
Specificity / Sensitivity
Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb recognizes endogenous levels of histone H3 protein only when phosphorylated at Thr3.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr3 of human histone H3 protein.
Confocal immunofluorescent analysis of HeLa cells using Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Raw 264.7 cells using Phospho-Histone H3 (Thr4) (D5G1I) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Peptide dot blot analysis demonstrating Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb specificity. Antibody binding to pre-coated phospho-histone H3 peptides is shown using Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb, Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377, Phospho-Histone H3 (Thr11) (C2A6) Rabbit mAb #9767, and Phospho-Histone H3 (Ser28) Antibody #9713. As expected, Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb only binds to phospho-histone H3 peptide when phosphorylated at Thr3.
Western blot analysis of extracts from 293T and Raw 264.7 cells, untreated (-) or treated with Nocodazole #2190 (100 ng/ml, 16 hr; +), using Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb (upper) and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower).
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells treated with Nocodazole #2190 (100 ng/ml) overnight, and either 5 μl of Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human α Satellite Repeat Primers #4486, and human D7Z1 satellite primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). 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 (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).
- Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
- Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
- Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-5.
- Cheung, P. et al. (2000) Cell 103, 263-71.
- Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73.
- Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
- Thorne, A.W. et al. (1990) Eur J Biochem 193, 701-13.
- Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
- Goto, H. et al. (1999) J Biol Chem 274, 25543-9.
- Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.
- Dai, J. et al. (2005) Genes Dev 19, 472-88.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
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For Research Use Only. Not For Use In Diagnostic Procedures.
DRAQ5 is a registered trademark of Biostatus Limited.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
Tween is a registered trademark of ICI Americas, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
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