It is intended to provide a cell-free Notch cleavage analysis method which is usable in screening a drug such as a Ϝ-secretase inhibitor efficacious in treating Alzheimer’s diseases, etc.; and the drug screening method described above. Namely, a cell-free Notch cleavage analysis method comprising the cell-free cleavage reaction step of contacting a crude membrane fraction of a cell expressing a Notch protein mutant with a test substance, and the detection step of detecting a fraction in the amino terminal side and a fraction in the carboxyl terminal side, which are formed by the intramembrane proteolysis (two-stage cleavage in a presumed transmembrane domain) of the Notch protein mutant in the cell-free cleavage reaction step as described above, to thereby analyze the effect of the test substance on the intramembrane proteolysis of the Notch protein mutant, wherein the Notch protein mutant has at least the presumed transmembrane domain of the Notch protein, lacks at least a part of the amino acid sequence in the amino terminal side of the presumed transmembrane domain, and has antibody recognition sites in both of the amino terminal side and the carboxyl terminal side of the presumed transmembrane domain.
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