Product Pathways - Cell Cycle / Checkpoint

Cell Cycle/Checkpoint Sampler Kit #9917

• pathway
• more info
• application references
• datasheet PDF
• MSDS PDF
• protocols

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  IF-P=Immunofluorescence (Paraffin)  IC=Immunocytochemistry  F=Flow Cytometry  E-P=ELISA (Peptide)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Dr=Drosophila  X=Xenopus  Z=Zebra Fish

Specificity / Sensitivity

Phospho-cdc2 (Tyr15) Antibody detects cdc2 and cdk2 only when phosphorylated at Tyr15 and does not cross-react with cdk4, cdk6 or cdk7. Phospho-Chk2 (Thr68) Antibody detects Chk2 only when phosphorylated at Thr68 and does not react with nonphosphorylated Chk2. Phospho-Chk1 (Ser345) Antibody detects Chk1 only when phosphorylated at Ser345 and does not cross-react with other proteins. Phospho-Rb (Ser795) and (Ser807/811) Antibodies detect Rb only when phosphorylated at the indicated sites and do not cross-react with Rb phosphorylated at other sites. Phospho-p53 (Ser15) 16G8 Monoclonal Antibody detects p53 only when phosphorylated at Ser15 and does not react with nonphosphorylated p53.

Western Blotting

Western blot analysis of extracts from UV or MMS treated COS cells, using Phospho-Chk1 (Ser345) Antibody #2341.

Western Blotting

Western blot analysis of extracts from UV or MMS treated C6 cells and HU treated HeLa cells, using Phospho-Chk1 (Ser345) Antibody #2341.

Western Blotting

Western blot analysis of extracts from UV treated MCF-7 cells and COS cells, using Phospho-Chk2 (Thr68) Antibody #2661.

Western Blotting

Western blot analysis of extracts from COS cells transfected with Wild-type Chk2, Chk2 (T68A), Chk2 (T26S28A) and Chk2 (S33S35A), using Phospho-Chk2 (Thr68) Antibody #2661.

Western Blotting

Western blot analysis of extracts from Saos cells, untreated, hydroxyurea-treated (G1/S) or nocodazole-treated (G2/M), using Phospho-cdc2 (Tyr15) Antibody #9111 or cdc2 Antibody #9112.

Western Blotting

Western blot analysis of cell extracts from hydroxyurea (20 mM) or UV treated MvlLu cells, using Phospho-p53 (Ser15) 16G8 mAb #9286.

Western Blotting

Western blot analysis of Rb phosphorylation in human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser795) Antibody #9301. Cells were synchronized for 24 hours, then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck.)

Western Blotting

Western blot analysis of Rb phosphorylation in human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser807/811) Antibody #9308. Cells were synchronized for 24 hours, then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck.)

IC-ABC

Immunocytochemical staining of untreated or hydroxyurea treated MvlLu cells, using Phospho-p53 (Ser15) 16G8 mAb #9286.

IF-IC

Immunofluorescent analysis of SK-N-MC cells, untreated (left) or hydroxyurea-treated (right), using Phospho-cdc2 (Tyr15) Antibody #9111.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides (KLH-coupled) corresponding to residues surrounding Tyr15 of human cdc2; Ser345 of human Chk1; Thr68 of human Chk2; and Ser795 and Ser807/811 of human Rb. Monoclonal antibody is produced by immunizing mice with a synthetic phospho-Ser15 peptide (KLH-coupled) corresponding to residues surrounding Ser15 of human p53. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The cell division cycle demands accuracy to avoid the accumulation of genetic damage. This process is controlled by molecular circuits called "checkpoints" that are common to all eukaryotic cells (1). Checkpoints monitor DNA integrity and cell growth prior to replication and division at the G1/S and G2/M transitions, respectively. The cdc2-cyclin B kinase is pivotal in regulating the G2/M transition (2,3). Cdc2 is phosphorylated at Thr14 and Tyr15 during G2-phase by the kinases Wee1 and Myt1, rendering it inactive. The tumor suppressor protein retinoblastoma (Rb) controls progression through the late G1 restriction point (R) and is a major regulator of the G1/S transition (4). During early and mid G1-phase, Rb binds to and represses the transcription factor E2F (5). The phosphorylation of Rb late in G1 by cdks induces Rb to dissociate from E2F, permitting the transcription of S-phase-promoting genes. Rb can be phosphorylated at multiple sites in vitro by cdc2, cdk2 and cdk4/6 (6-8). DNA damage triggers both the G2/M and the G1/S checkpoints. DNA damage activates the DNA-PK/ATM/ ATR kinases, which phosphorylate Chk at Ser345 (9), Chk2 at Thr68 (10) and p53 (11). The Chk kinases inactivate cdc25 via phosphorylation at Ser216, blocking the activation of cdc2.

Description: The Cell Cycle/Checkpoint Sampler Kit provides a fast and economical means to evaluate the activation status of multiple members of cell cycle/ checkpoint signaling pathways, including phospho-cdc2 (Tyr15), phospho-Chk1 (Ser345), phospho-Chk2 (Thr68), phospho-Rb (Ser795, Ser807/811) and phospho-p53 (Ser15). The kit contains enough primary and secondary antibodies to perform four mini-blot experiments.

1. Nurse, P. (1997) Cell 91, 865-7.
2. Norbury, C. and Nurse, P. (1992) Annu Rev Biochem 61, 441-70.
3. Watanabe, N. et al. (1995) EMBO J. 14, 1878-1891.
4. Sherr, C.J. (1996) Science 274, 1672-1677.
5. Dyson, N. (1998) Genes Dev. 12, 2245-2262.
6. Kitagawa, M. et al. (1996) EMBO J. 15, 7060-7069.
7. Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-761.
8. Harbour, J.W. et al. (1999) Cell 98, 859-869.
9. Zhao, H. and Piwnica-Worms, H. (2001) Mol. Cell Biol. 21, 4129-4139.
10. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
11. Tibbetts, R.S. et al. (1999) Genes Dev. 13, 152-157.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

• 9112 cdc2 Antibody
• 9289 Phospho-p53 (Ser37) Antibody
• 9307 Phospho-Rb (Ser780) Antibody
• 9919 Phospho-p53 Antibody Sampler Kit
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7076 Anti-mouse IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide