Identification of Active Caspases Using Affinity-Based Probes

Small-molecule inhibitors of caspases can be modified with moieties such as biotin or fluorescent molecules. After the inhibitor molecule has bound to an active caspase, the caspase itself becomes labeled and can be isolated using affinity purification. This protocol describes the use of the biotinylated pan-caspase inhibitor VAD-FMK and streptavidin beads to isolate active caspases. These caspases are then separated by gel electrophoresis and identified with caspase-specific antibodies using western blotting techniques. Other caspase inhibitors bound with biotin or other labels can be substituted in this assay; labeled inhibitors are available commercially as either pan-caspase or caspase-specific probes.