Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Phospho-CENP-A (Ser7) Antibody #2187

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC H (Mk) Endogenous 17 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

Phospho-CENP-A (Ser7) Antibody detects endogenous levels of human CENP-A protein only when phosphorylated on Ser7. This antibody does not cross-react with other histone proteins, including Histone H3.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser7 of human CENP-A protein. Antibodies are purified by peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells arrested in S phase or mitosis using Phospho-CENP-A (Ser7) Antibody (upper panel) or CENP-A Antibody #2186 (lower panel). S phase cells were treated for 12 hours with thymidine (2 mM), rinsed three times, released into normal growth medium for 10 hours and then treated an additional 12 hours with thymidine before harvesting. Mitotic cells were treated for 12 hours with thymidine, rinsed three times and then treated for 16 hours with paxitaxol (500 nM final).

IF-IC

IF-IC

Confocal immunofluorescent analysis of a mitotic HeLa cell using Phospho-CENP-A (Ser7) Antibody (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red). Phospho-CENP-A signal is localized to bright spots in the metaphase plate. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Background

Modulation of chromatin structure plays a critical role in the regulation of transcription and replication of the eukaryotic genome. The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). CENP-A, also known as the chromatin-associated protein CSE4 (capping-enzyme suppressor 4-p), is an essential histone H3 variant that replaces canonical histone H3 in centromeric heterochromatin (2). The greatest divergence between CENP-A and canonical histone H3 occurs in the amino-terminal tail of the protein, which binds linker DNA between nucleosomes and facilitates proper folding of centromeric heterochromatin (3). The amino-terminal tail of CENP-A is also required for recruitment of other centromeric proteins (CENP-C, hSMC1, hZW10), proper kinetochore assembly and chromosome segregation during mitosis (4). Additional sequence divergence in the histone fold domain is responsible for correct targeting of CENP-A to the centromere (5). Many of the functions of CENP-A are regulated by phosphorylation (6,7). Aurora A-dependent phosphorylation of CENP-A on Ser7 during prophase is required for proper targeting of Aurora B to the inner centromere in prometaphase, proper kinetochore/microtubule attachment and proper alignment of chromosomes during mitosis (6).

  1. Jin, J. et al. (2005) Trends Biochem Sci 30, 680-7.
  2. Ausió, J. (2006) Brief Funct Genomic Proteomic 5, 228-43.
  3. Heit, R. et al. (2006) Biochem Cell Biol 84, 605-18.
  4. Van Hooser, A.A. et al. (2001) J Cell Sci 114, 3529-42.
  5. Black, B.E. et al. (2004) Nature 430, 578-82.
  6. Kunitoku, N. et al. (2003) Dev Cell 5, 853-64.
  7. Zeitlin, S.G. et al. (2001) J Cell Biol 155, 1147-57.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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