Precision plus molecular weight marker

Yellow field pea flour (~20% protein content) was provided by AGT Foods (Regina, SK, Canada) and commercial pea protein isolate (cPPI, 81.2% protein, 3.86% ash), PURIS™ Pea Protein, was provided by Puris Foods (Minneapolis, MN, USA). Commercial whey protein isolate (cWPI, 94.6% protein, 4.10% ash), BiPro^®, was provided by Agropur Ingredients (Eden Prairie, MN, USA). Defatted soy flour (~53% protein) and commercial soy protein isolate (cSPI, ~90.7% protein), ProFam^® 974, were provided by Archer Daniels Midland (ADM) (Decatur, IL, USA). All aforementioned samples were stored at −20 °C prior to usage. SnakeSkin™ dialysis tubing (3.5 kDa cut off) and Sudan Red 7B were purchased from Thermo Fisher Scientific™ (Waltham, MA, USA). Criterion™ TGX™ 4–20% precast gels, Laemmli 4X loading buffer, Imperial™ Protein Stain, 10X Tris/Glycine/SDS running buffer and Precision Plus molecular weight marker were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Vivaflow^® membrane ultrafiltration crossflow cassettes (3 kDa MWCO) were purchased from Sartorius™ (Gottingen, Germany). All other chemical reagents and supplies were purchased from Thermo Fisher Scientific and Sigma-Aldrich.

Antibodies used for western blotting were as follows: anti-Flag M2 (1:10000, Sigma-Aldrich, St. Louis, MO, USA); anti-NSF (1:500, Cell Signaling, Danvers, MA, USA); anti-LRRK2 (1:1000, C41-2, Abcam, Cambridge, UK); anti-Synaptobrevin, anti-synaptophysin and anti-Synaptotagmin 1 (1:1000, Synaptic System, Göttingen, Germany).
Between 10 and 20 μg of protein samples were dissolved in 4–20 % Tris-glycine polyacrylamide gels (Biorad) in SDS/Tris-glycine running buffer. Precision Plus molecular weight markers (Biorad) were used for size estimation. Solubilized proteins were then transferred to polyvinylidenedifluoride (PVDF) membranes in transfer buffer containing 10 % methanol. The PVDF sheets were blocked in Tris-buffered saline plus 0.1 % Triton (TBS-T) plus 5 % nonfat dry milk for 1 h at 4 °C and then incubated overnight at 4 °C with primary antibody in TBS-T plus 5 % non-fat dry milk. The PVDF membranes were washed in TBS-T (3 × 10 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated anti-mouse IgG. Blots were then washed in TBS-T (4 × 10 min) at RT and rinsed in TBS, and immunoreactive proteins were visualized using enhanced chemiluminescence plus (ECL+, GE Healthcare, Waukesha, WI, USA). Densitometric analysis was carried out using Image J software.