Cell Cycle Phase Determination Antibody Sampler Kit #17498
Product Information
Kit Usage Information
Protocols
- 4539: Western Blotting, Immunoprecipitation (Agarose), Immunofluorescence, Flow
- 7074: Western Blotting
- 8064: Western Blotting, Immunoprecipitation (Agarose), Immunofluorescence
- 12231: Western Blotting, Immunoprecipitation (Magnetic), Immunofluorescence, Flow
- 20808: Western Blotting
- 28755: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), Immunofluorescence, Flow
- 52508: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), Immunofluorescence
- 53348: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Leica® Bond™), Immunohistochemistry (Paraffin), Immunofluorescence, Flow, ChIP Magnetic
- 91500: Western Blotting
Product Description
The Cell Cycle Phase Determination Antibody Sampler Kit provides an economical means of detecting total proteins or post-translational modifications present in cells at various phases of the cell cycle. Geminin is degraded in G1 phase, while CDT1 is degraded in S, G2, and M phases. Thymidine Kinase 1 accumulates in G1 phase, peaks in S phase, and is degraded before cell division. Phospho-Histone H3 (Ser10) is present only in M phase, while Phospho-cdc2 (Tyr15) is absent in M phase. Cyclins A2, B1, and E1 peak at G2 phase, late G2/M phase, and late G1/early S phase, respectively. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.
Background
The entry of eukaryotic cells into mitosis is regulated by cdc2/CDK1 kinase activation, a process controlled at several steps including cyclin B1 nuclear accumulation and binding, and phosphorylation of cdc2/CDK1 at Thr161 (1). At the end of mitosis, cyclin B1 is targeted for degradation by the anaphase-promoting complex (APC), allowing for cell cycle progression (2). A critical regulatory step in activating cdc2 during progression into mitosis is dephosphorylation of cdc2/CDK1 at Thr14 and Tyr15 (3).
Phosphorylation of Histone H3 at Ser10 is tightly correlated with chromosome condensation during both mitosis and meiosis (4).
Overcoming the G1/S checkpoint to commence DNA replication requires cyclin E, traversing the G2/M checkpoint to initiate mitosis requires cyclin B, and cyclin A is required for both S-phase and M-phase (5). Cyclin A availability is apparently the rate-limiting step for entry into mitosis, and cyclin A is required for completion of prophase (6).
Thymidine kinases play a critical role in generating the DNA synthetic precursor deoxythymidine triphosphate (dTTP). Cytoplasmic thymidine kinase 1 (TK1) expression and activity are regulated in a cell cycle-dependent manner, accumulating during G1-phase to peak levels in S-phase before being degraded prior to cell division (7).
The initiation of S phase begins with the formation of the pre-replication complex (pre-RC) in late mitosis/early G1 phase. CDT1 and cdc6 bind to the origin of DNA replication, which allows binding of the MCM2-7 complex. In order to ensure that replication occurs only once per cell cycle, geminin inhibits and destabilizes CDT1 during the S, G2 and M phases. At the metaphase/anaphase transition, geminin is degraded by the anaphase-promoting complex (APC) allowing for the formation of new pre-RC (8).
Phosphorylation of Histone H3 at Ser10 is tightly correlated with chromosome condensation during both mitosis and meiosis (4).
Overcoming the G1/S checkpoint to commence DNA replication requires cyclin E, traversing the G2/M checkpoint to initiate mitosis requires cyclin B, and cyclin A is required for both S-phase and M-phase (5). Cyclin A availability is apparently the rate-limiting step for entry into mitosis, and cyclin A is required for completion of prophase (6).
Thymidine kinases play a critical role in generating the DNA synthetic precursor deoxythymidine triphosphate (dTTP). Cytoplasmic thymidine kinase 1 (TK1) expression and activity are regulated in a cell cycle-dependent manner, accumulating during G1-phase to peak levels in S-phase before being degraded prior to cell division (7).
The initiation of S phase begins with the formation of the pre-replication complex (pre-RC) in late mitosis/early G1 phase. CDT1 and cdc6 bind to the origin of DNA replication, which allows binding of the MCM2-7 complex. In order to ensure that replication occurs only once per cell cycle, geminin inhibits and destabilizes CDT1 during the S, G2 and M phases. At the metaphase/anaphase transition, geminin is degraded by the anaphase-promoting complex (APC) allowing for the formation of new pre-RC (8).
- Atherton-Fessler, S. et al. (1994) Mol Biol Cell 5, 989-1001.
- Gong, D. and Ferrell, J.E. (2010) Mol Biol Cell 21, 3149-61.
- Norbury, C. et al. (1991) EMBO J 10, 3321-9.
- Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
- Pagano, M. et al. (1992) EMBO J 11, 961-71.
- Furuno, N. et al. (1999) J Cell Biol 147, 295-306.
- Munch-Petersen, B. (2010) Nucleosides Nucleotides Nucleic Acids 29, 363-9.
- Caillat, C. and Perrakis, A. (2012) Subcell Biochem 62, 71-87.
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