Patent ID: 7700375

Claim:
An assay method for detecting target proteins having sterically unhindered sulfhydryl groups, said assay comprising: generating: 1) at least a first dimaleimide molecule, said dimaleimide molecule having the following formula: wherein each R or R′, independently, is R a or OR a and R a is H, C 1 -C 4 alkyl, CH 2 CO 2 H, CH 2 CH 2 OH or CN and Ar is a rigid aromatic skeleton and selected from the group consisting of fluorescein, rhodamine, eosin, thionine, safranin, fluorescent compounds having naphthyl groups and coumarin; wherein the substituent maleimide groups are positioned around the aromatic core in such a manner that they are separated by 5, 10 or 15 Å, conferring complementarity for reaction of both maleimide groups of the fluorogen with the sulthydryl groups of said target protein having sterically unhindered sulfhydryl groups; or said dimaleimide molecule has the following formula: wherein each R or R′, independently, is R a or OR a and R a is H, C 1 -C 4 alkyl, CH 2 CO 2 H, CH 2 CH 2 OH or CN and Ar is a rigid aromatic skeleton comprising one aromatic ring or two to four fused aromatic rings selected from the group consisting of phenyl, naphthyl, anthracene, fluorene, pyridine, pyrimidine, purine, and indole, X is a spacer sequence that includes C 1 -C 4 alkyl, OCH 2 CH 2 O, NHCO(C 1 -C 4 CH 2 alkyl)NHCO, CONH(C 1 -C 4 CH 2 alkyl)NHCO, NHCO(C 1 -C 4 CH 2 alkyl)CONH, CONH(C 1 -C 4 CH 2 alkyl)CONH, and Fl is a fluorophore selected from the group consisting of fluorescein, rhodamine, eosin, thionine, safranin, and coumarin; wherein the substituent maleimide groups are also positioned around the aromatic core in such a manner that they are separated by 5, 10 or 15 Å, conferring complementarity for reaction of both maleimide groups of the fluorogen with the sulfhydryl groups of said target protein having sterically unhindered sulfhydryl groups 2) at least a target protein having sterically unhindered sulfhydryl groups, or a target protein linked or fused to a protein of interest; or 3) nucleic acid molecules that code for said target protein or a target protein fused to a protein of interest as defined in 2) and subsequently allowing said nucleic acid molecules to produce their coded products; then, (A) allowing reaction of said target protein having sterically unhindered sulfhydryl groups or a target protein fused to a protein of interest with said dimaleimide molecule; and (B) detecting a a fluorescent signal from said complex of target protein or a target protein fused to a protein of interest and dimaleimide molecule.