Product Pathways - Cell Cycle / Checkpoint
Cell Cycle/Checkpoint Antibody Sampler Kit #9917
|Kit Includes||Quantity||Applications||Reactivity||MW (kDa)||Isotype|
|Phospho-cdc2 (Tyr15) Antibody #9111||40 µl||W IP||H M R Mk Dm X||34||Rabbit|
|Phospho-Chk1 (Ser345) (133D3) Rabbit mAb #2348||40 µl||W IF-IC F||H M R Mk||56||Rabbit IgG|
|Phospho-Chk2 (Thr68) Antibody #2661||40 µl||W IP IF-IC F||H Mk||62||Rabbit|
|Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb #8516||40 µl||W IP IHC-P IF-IC F||H M R Mk||110||Rabbit IgG|
|Phospho-Rb (Ser795) Antibody #9301||40 µl||W IP||H R Mk||110||Rabbit|
|Phospho-p53 (Ser15) Antibody #9284||40 µl||W IP IF-IC ChIP||H M R Mk (Mi) (B) (Pg)||53||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
Reactivity Key: H=Human M=Mouse R=Rat Mk=Monkey Mi=Mink Dm=D. melanogaster X=Xenopus B=Bovine Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
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) Antibody detects p53 only when phosphorylated at Ser15 and does not react with nonphosphorylated p53.
Western blot analysis of extracts from UV or MMS treated COS cells, using Phospho-Chk1 (Ser345) Antibody #2341.
Western blot analysis of extracts from HeLa, COS, NIH/3T3 and C6 cells, untreated or UV-treated, using Phospho-Chk1 (Ser345) (133D30) Rabbit mAb #2348.
Western blot analysis of extracts from Cos cells, untreated or UV-treated (100 mJ/cm2, 1 hr recovery), using Phospho-Chk2 (Thr68) Antibody #2661.
Western blot analysis of extracts from WI-38 cells, serum-starved for 3 days (-) or serum-starved for 3 days followed by treatment with 10% serum for 2 days (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb #8516.
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 blot analysis of extracts from Mv1Lu cells treated with UV or hydroxyurea (20 mM) for the indicated times, using Phospho-p53 (Ser15) Antibody #9284.
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.)
The Cell Cycle/Checkpoint Antibody Sampler Kit provides a fast and economical means of evaluating multiple proteins involved in the cell cyle and checkpoint control. The kit contains enough primary and secondary antibody to perform four Western blot experiments.
Source / Purification
Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr15 of human cdc2; Thr68 of human Chk2; Ser795 and Ser15 of human p53. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser807/811 of human Rb protein and residues surrounding Ser345 of human Chk1.
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-phase by CDKs induces Rb to dissociate from E2F, permitting the transcription of S-phase-promoting genes. In vitro, Rb can be phosphorylated at multiple sites 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.
- Nurse, P. (1997) Cell 91, 865-7.
- Norbury, C. and Nurse, P. (1992) Annu Rev Biochem 61, 441-70.
- Watanabe, N. et al. (1995) EMBO J 14, 1878-91.
- Sherr, C.J. (1996) Science 274, 1672-7.
- Dyson, N. (1998) Genes Dev 12, 2245-62.
- Kitagawa, M. et al. (1996) EMBO J 15, 7060-9.
- Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-61.
- Harbour, J.W. et al. (1999) Cell 98, 859-69.
- Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39.
- Matsuoka, S. et al. (2000) Proc Natl Acad Sci USA 97, 10389-94.
- Tibbetts, R.S. et al. (1999) Genes Dev 13, 152-7.
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For Research Use Only. Not For Use In Diagnostic Procedures.