A substrate-optimized electrophoretic mobility shift assay for ADAM12

Abstract

ADAM12 belongs to the A disintegrin and metalloprotease (ADAM) family of secreted sheddases activating extracellular growth factors such as epidermal growth factor receptor (EGFR) ligands and tumor necrosis factor-alpha (TNF-α). ADAM proteases, most notably ADAM17 (TNF-α-converting enzyme), have long been investigated as pharmaceutical drug targets; however, due to lack of potency and in vivo side effects, none of the small-molecule inhibitors discovered so far has made it beyond clinical testing. Ongoing research on novel selective inhibitors of ADAMs requires reliable biochemical assays to validate molecular probes from large-scale screening efforts. Here we describe an electrophoretic mobility shift assay for ADAM12 based on the identification of an optimized peptide substrate that is characterized by excellent performance and reproducibility.

Keywords: ADAM12; Fluorimetric assay; Metalloprotease; Mobility shift assay; Peptide array; Small-molecule inhibitor.