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For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala35 of human CDC20 protein. Antibodies are purified by protein A and peptide affinity chromatography.
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.
CDC20 binds to and activates the anaphase-promoting complex (APC) during mitosis and G1 phase of the cell cycle (12). Moreover, CDC20 is necessary for ubiquitin ligase activity of the APC/cyclosome (APC/C). In metaphase MAD2L1 inactivates the CDC20-APC/C complex, while in anaphase this inhibition is lost and CDC20-APC/C degrades its substrates (13). p53 and p21 suppress expression of CDC20 upon genotoxic stresses and ectopic introduction of p53. siRNA mediated knock-down of CDC20 in cancer cells leads to attenuated cell growth and induces G(2)/M arrest, suggesting that CDC20 is a possible therapeutic target of cancer (14). Organization of neuronal circuits requires presynaptic axonal differentiation and synapse formation. CDC20-APC regulates presynaptic differentiation in postmitotic neurons by triggering the required degradation of the transcription factor NeuroD2 (15).
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