Titlebook: Checkpoint Controls and Cancer; Volume 2: Activation Axel H. Schönthal Book 2004 Humana Press 2004

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Assaying Cdc25 Phosphatase Activity25 proteins produced in . or insect cells, a fluorimetric assay using fluorescein diphosphate (FDP) as a substrate is recommended. To analyze endogenous Cdc25 phosphatase activities of immuno-precipitates from total cellular extracts, the physiological substrate Cdk1/cyclin B1 is most sensitive.

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Analyzing the Regulation and Function of ATMned into an inducible EBV-based vector (pMEP4) and its expression analyzed in a stably transfected cell line. ATM protein induction is monitored by immunoblotting with antibodies against both ATM and a FLAG sequence tag in the recombinant protein. Extracts from irradiated cells are immunoprecipitate

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Analysis of the Spindle-Assembly Checkpoint in HeLa Cellsachment or spindle tension. Here, we detail procedures for the analysis of the spindle-assembly checkpoint in adherent mammalian cells. These techniques focus on pharmacological approaches and immunofluorescence microscopy to verify the state of spindle assembly, kinetochore attachment of microtubul

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Functional Analysis of the Spindle-Checkpoint Proteins Using an In Vitro Ubiquitination Assayy attached to the mitotic spindle, this checkpoint blocks the activity of a large ubiquitin protein ligase complex, called the anaphase-promoting complex (APC) or cyclosome. This chapter describes the detailed protocols of an in vitro ubiquitination assay reconstituted with purified APC, cofactors,