Practical considerations in the use of affinity labelling reagents

Despite the original intent to design affinity labelling reagents with some selectivity, YVK(bio)D-aomk and Z-EK(bio)D-aomk lack selectivity for their originally intended caspase targets under the conditions commonly utilized. YVK(bio)D-aomk has been successfully utilized to label the caspases that are present in extracts from apoptotic cells, including the proteases that cleave poly(ADP-ribose) polymerase and the lamins (42); and Z-EK(bio)D-aomk readily reacts with recombinant caspases-1 and -2 as well as caspases-3 and -6 (18). Although these reagents might show some selectivity for various caspases during brief labelling at low ligand concentrations, prolonged incubation with high concentrations of these reagents easily compensates for poor affinity, as demonstrated by Margolin et al. (34).

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