ADAM17 Activity Assay

ADAM17 activity in cell lysates was determined using the InnoZyme TACE Activity Kit (Calbiochem; CBA042) according to the manufacturer's protocol. Cell were lysed using the CytoBuster Protein Extraction Reagent (EMD Millipore; 71009) followed by centrifugation at 15,000 rpm for 8 minutes. Protein concentration was quantified in the filtered supernatant using a NanoDrop spectrophotometer. Purified recombinant human TACE was used as a positive control. The lysates were incubated in an anti-human ADAM17 antibody precoated 96-well plate for 1 hour, followed by five washing steps. Detection solution containing an internally quenched fluorescent substrate was added for 4 hours and fluorescence was measured at an excitation wavelength of 324 nm and emission wavelength of 400 nm using a TECAN Infinite 200 PRO spectrophotometer. The level of fluorescence was directly related to the enzyme activity.