title: 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.
authors.affiliation: Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. Electronic address: kotzsch@bcube-dresden.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kotzsch
authors.affiliation: Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Tine
authors.initials: T
authors.lastname: Skovgaard
authors.affiliation: Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Uwe
authors.initials: U
authors.lastname: Buus
authors.affiliation: Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Andersen
authors.affiliation: Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Kanchan
authors.initials: K
authors.lastname: Devkota
authors.affiliation: Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Berthelsen
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000072199:ADAM12 Protein; D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results
chemical_list: D006877:Hydroxamic Acids; D008565:Membrane Proteins; D010455:Peptides; D011480:Protease Inhibitors; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human
keywords: ADAM12; Fluorimetric assay; Metalloprotease; Mobility shift assay; Peptide array; Small-molecule inhibitor