GCase activity by MDW941 probe method

These methods apply specifically to sample preparation at Pfizer for assessment of brain and liver tissue for GCase activity by the MDW941 probe method. Lysates were prepared by excision of cortex from the frozen brain hemisphere and from tissue obtained from the frozen liver lobe. Tissues were homogenized in 10 volumes of GCase lysis buffer containing 0.25% Triton X-100 using Qiagen Tissuelyser at 25 Hz for 4 min x 2. Samples were then sonicated with a Branson Ultrasonics 450 Digital Sonifier (Branson) for 10 seconds at 80 watts at 20 kHz and diluted to 2 mg/mL with lysis buffer. The GCase activity probe MDW941 was added to the samples to a final concentration of 25 nM probe, 5mM citric acid. Lysate and MDW941 probe were incubated at 37°C for 2 hours. Samples were centrifuged at 21,000 x g for 2 minutes to remove acid precipitates, the supernatant was retained. NuPAGE LDS (4X) sample buffer (ThermoFisher) containing NuPAGE Sample Reducing Agent (dithiothreitol) was added and the mixtures were incubated at 70°C for 10 min. Protein concentration was determined by BCA (Pierce). 30 μg of protein lysate samples were loaded onto a 4–12% gel NuPAGE (ThermoFisher) with TAMRA labelled fluorescent ladder and probe labelled 5 ng recombinant GCase and run at 150 V in MES buffer until the dye front reached the bottom of the gel. A 20% methanol solution was used to wash away unbound probe.

Gels were visualized using a GE Typhoon at 532 nm excitation and 575 nM Long Pass Filter emission, PMT 1000. Bands were quantified for relative fluorescence units by ImageQuant software (GE). Two-Way ANOVA tests were performed for statistical analysis to understand the effect of genotype and age. When significance was identified by the Two-Way ANOVA test, Bonferroni post hoc tests were used to understand the significance of the individual comparisons. Individual data points with mean and standard error of the mean are represented on each graph.