Bromophenol blue dye

The method described by Lee et al. (28 (link)) was adopted for gustatory assay. Adult flies were reared for 20 or 40 days in media supplemented with 10% PJ or 200 μM RV. A basal media control was also maintained. To perform a feeding assay, after starving the flies for 2 h, 30 male or female flies from each experimental group were transferred into the vials containing the specific diets with bromophenol blue dye (0.05% wt/vol) (Sigma-Aldrich, St. Louis, MO, USA). After 10 min of feeding, the fed flies were etherized, washed with phosphate-buffered saline (PBS), and homogenized in 1 ml of distilled water. The absorbance of 100 times diluted supernatant was measured at 595 nm using a spectrophotometer (Bio-Rad, CA, USA).

Electrophoresis was performed with a 15% SDS polyacrylamide slab minigel and a Tris-glycine buffer system (SDS-PAGE). Human recombinant alpha-synuclein (600 ng) was loaded and subjected to 20 mA current until the bromophenol blue dye (Sigma-Aldrich) approached the end of the gel. Proteins were transferred at 150 mA for 4 h onto a 0.1 μm pore-size nitrocellulose membrane using a TRIS-glycine-methanol buffer. The membrane was washed in 0.1% Tween-20/PBS and blocked for 1 h with 5% skimmed milk (SM) (Sucofin, TSI GmbH & Co. KG, Zeven, Germany)/PBS. After washing, the membrane was cut into strips and incubated overnight at 4°C with 2% SM/PBS with 1:500 rabbit anti-alpha/beta/gamma-synuclein IgG (FL-140) or 1:1000 mouse syn211 anti-alpha-synuclein IgG antibody. After washing and blocking, strips were incubated with anti-rabbit or anti-mouse IgG-alkaline phosphatase (AP) at 1:5000 for 2 h in 2% SM/PBS. The immunoblot was visualized using BCIP (5-bromo-4-chloro-3-indolyl-phosphate) with NBT (nitro blue tetrazolium) (Promega, Mannheim, Germany) for detection of AP activity.