Sensolyte 520 ide activity assay kit

Liver IDE activity measurement was performed using the SensoLyte 520 IDE Activity Assay Kit (AnaSpec, Fremont, CA, United States, cat. AS-72231) following the manufacturer’s instructions. The total IDE activity was calculated as previously described (Kurauti et al., 2016 (link)) and normalized per μg of total protein, which was determined using Bradford reagent. The kinetic concentration of 5-FAM was also normalized per μg of total protein.

IDE activity was assessed in the liver and muscle protein lysates using a SensoLyte 520 IDE Activity Assay Kit (catalog #AS-72231, AnaSpec, Fremont, CA, USA) according to the manufacturer's instructions. Activity was expressed as RFU/mg protein over a time course of 60 min, with sampling every 5 min. Reaction rates were calculated as the slope of the linear regression between 10 and 50 min.

Liver IDE activity was measured using a SensoLyte 520 IDE Activity Assay Kit (Catalog #AS-72231; AnaSpec, Fremont, Canada) by following the manufacturer’s instructions. Total IDE activity was calculated as described previously [15 (link)] and normalized per μg of protein content, as determined using a Bio-Rad Protein Assay Dye Reagent Concentrate (Catalog #5000006, BioRad, Hercules, CA, USA).

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.

IDE activity was measured in liver samples by SensoLyte 520 IDE Activity Assay Kit (AnaSpec, Fremont, Canada; cat No. AS‐72231) following the manufacturer’s instructions. Total IDE activity was calculated as previously described^{15 (link)} and was normalized per μg of total protein content determined using the Bradford reagent.

Analysis of IDE activity was performed by a fluorometric assay with SensoLyte 520 IDE activity assay kit (AS-72231, Anaspec, Fremont, CA, USA). Protein extraction was carried out by sonicating individual retinas in the commercial assay buffer. The activity of IDE was assayed following the manufacturer’s instructions, and fluorescence was detected with Varioskan LUX (ThermoFisher Scientific). IDE activity was normalized to total protein in the sample as quantified with Pierce BCA assay kit (ThermoFisher Scientific). IDE activity in an individual mouse corresponds to the mean value of both retinas.