Cell Cycle Checkpoint BioAssay™ Sample Kit (cdc2 (Tyr15), Chk1 (Ser345), Chk2 (Thr68), Rb (Ser795, Ser807/811), p53 (Ser15))

Kits and Assays / Kits-Molecular Biology, BioAssay™

-20°C

All antibodies are supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, BSA, 50% glycerol.

Synthetic peptides (KLH coupled) corresponding to residues surrounding PhosphorylatedTyr15 of human cdc2; Phosphorylated Ser345 of human Chk1; Phosphorylated Thr68 of human Chk2; and Phosphorylated Ser795 and Phosphorylated Ser807/811 of human Rb. Mab is produced by immunizing mice with a synthetic Phosphorylated Ser15 peptide (KLH coupled) corresponding to residues surrounding Phosphorylated Ser15 of human p53.

Recognizes cdc2 Phosphorylated (Tyr15), Chk1 Phosphorylated (Ser345), Chk2 Phosphorylated (Thr68), Rb Phosphorylated (Ser795, Ser807/811) and p53 Phosphorylated (Ser15).

The cell-replication cycle demands great accuracy in order 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 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 Chk1 at Ser345, Chk2 at Thr68 and the tumor suppressor protein p53 (9–11). The Chk kinases inactivate cdc25 via phosphorylation at Ser216, blocking the activation of cdc2 and the transition into M-phase. The phosphorylation of p53 at Ser15 enables its dissociation from MDM2, activating its DNA binding activity. The Cell Cycle/Checkpoint Sampler Kit provides a fast and economical means to evaluate the activation status of multiple members of the cell cycle/ checkpoint signal, including cdc2 (Tyr15), Chk1 (Ser345), Chk2 (Thr68), Rb (Ser795, Ser807/811) and p53 (Ser15). The kit contains enough primary and secondary antibodies to perform four mini-blot experiments.

Kit Components:
C2612-01: cdc2 Phosphorylated (Tyr15) Pab 1x20ul Rabbit IgG 34kD
C2612-02: Chk1 Phosphorylated (Ser345) Pab 1x20ul Rabbit IgG 56kD
C2612-03: Chk2 Phosphorylated (Thr68) Pab 1x20ul Rabbit IgG 62kD
C2612-04: Rb Phosphorylated (Ser795) Pab 1x20ul Rabbit IgG 110kD
C2612-05: Rb Phosphorylated (Ser807/811) Pab 1x20ul Rabbit IgG 110kD
C2612-06: p53 Phosphorylated (Ser15) Mab 1x20ul Mouse IgG 53kD
C2612-07: IgG (HRP) 1x100ul Pab GtxRb


Recommended Dilution:
Western Blot: 1:1000
Optimal dilutions to be determined by the researcher.

Storage and Stability:
May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer