© 2004 American Chemical Society
When it comes to enzymes, protein abundance does not necessarily translate into protein activity: Post-translational modifications such as phosphorylation can dramatically alter enzyme function. Researchers traditionally have overcome this discrepancy with activity-based protein profiling (ABPP), which screens enzyme activity using probes that bind covalently to active sites.
Most ABPP experiments employ in-gel fluorescence scanning to detect probe-labeled enzymes, but this method has several problems, including slow throughput, limited sensitivity, and poor resolution, says Benjamin Cravatt of the Scripps Research Institute, La Jolla, Calif. Cravatt and colleagues have developed a novel microarray-based approach that they say circumvents these issues.1
In Cravatt's method, enzymes are labeled with fluorescent-tagged probes and incubated with a glass slide containing spatially segregated spots of antibodies targeting a particular enzyme class. The slide is washed and the active, probe-labeled proteases that remain bound are quantified by fluorescence scanning of the microarray....