Fig. 2. Biochemical evaluation of second generation quenched fluorescent activity based probes. (a) Direct labeling of recombinant caspase-3 with qABPs. The indicated probes were incubated at various concentrations with recombinant caspase-3 for one hour, the reaction was stopped, separated by SDS PAGE and the gel was scanned for fluorescence. Samples marked with ⁺ represent pretreatment with the caspase/legumain inhibitor AB46 peptide which was added 30 min prior to probe. (b) Direct labeling of legumain in RAW cell lysate. Similar labeling conditions as in (a) were used. Samples were pretreated with the following inhibitors for 30 min prior to probe labeling: caspase-3 (AB46 peptide), a, cathepsin GB111-NH₂, b, or legumain-compound 5, c. (c) Quenching efficiency. Fluorescence of increasing concentration of qABPs 21 and 22 was measured relative to the non-quenched AB46-Cy5 with an IVIS scanner, excitation/emission 640/695–770 nm. Left panel, a graph showing reduced fluorescence. Right panel, a fluorescent picture of the dilution plate. Both probes were found highly quenched, 21 50 fold and 22 150 fold. (d) Time course of caspase-3 labeling in intact cells. Intact MM1s and KMS11 cells were treated with etoposide for indicated times, 22 was added for the last three hours. The caspase inhibitor ZVAD-FMK was added 9 or 3 hours prior to 22. Protein lysates were loaded and resolved by SDS-PAGE, followed by scanning on a flatbed laser scanner for Cy5 fluorescence. (e) Samples of MM1s cells from d were subjected to western blotting and reacted with a cleaved caspase-3 antibody.