Fluorometric IDE Activity Assay

Activity of IDE was measured as reported previously^{68 (link)} using fluorometric SensoLyte® 520 IDE activity assay kit (Cat # AS-72231, AnaSpec, Inc, Fremont, CA, USA) following the manufacturer’s protocol. Briefly, brain frontal cortical tissue extracts were homogenized in cold assay buffer (AnaSpec, Inc, Fremont, CA, USA) supplemented with complete protease inhibitor cocktail tablet (Cat # 11836170001, Sigma Aldrich) plus 0.3 mM PMSF. Homogenates were kept on ice for 30 min, followed by centrifugation at 10,000 X g for 15 min at 4 °C. Protein concentration was measured in the supernatants. Subsequently, enzyme reaction was set up by adding 50 µl of tissue lysates containing 100 µg protein/well in a 96-well black opaque plate. The enzymatic reaction was started by adding 50 µl fluorogenic substrate into each well. The plate was shaken for 30 s, and the reaction was incubated at 37 °C for 60 min in the dark. As a positive control, purified recombinant human IDE provided in the kit was used. The fluorescence intensity was measured at Ex/Em = 490 nm/520 nm using GloMax plate reader (Promega, Madison WI). The fluorescence readings from the wells containing the assay buffer without tissue lysates were used as a background fluorescence. The background reading was subtracted from the reading of the samples, and results are expressed as fluorescence intensity/µg protein.