反应详情

A protein gel was run to visualize the fractionated protein. 10-20 μl of each sample was loaded onto a pre-cast 10% Bis-Tris SDS-PAGE gel (Invitrogen, cat# NP0302BOX) for electrophoresis using the XCell SureLock™ Mini-Cell (Invitrogen, cat# EI0001). The samples were run for 35 minutes at 200V in MES-SDS running buffer (Invitrogen, cat# NP0002). The gel was stained with Coomassie Blue R-250 (Bio-Rad, cat#161-0436), as illustrated in FIG. 3. Fractions containing gamma-zein/YFP samples were pooled and transferred into a Millipore Spin column with 10 kDa MWCO, and centrifuged at 4,000 rpm for 20 minutes at 4° C. using a bench top Eppendorf centrifuge (Model 5810R). After centrifugation, sterile PBS solution was added up to 15 ml for buffer exchange. This spin and diafiltration process was repeated three times. The pooled gamma-zein/YFP samples were transferred into a SnakeSkin™ pleated dialysis tubing (Thermo Scientific, cat#68100) with a 10 kDa MWCO, and dialyzed against 2 L of PBS at 4° C. overnight to remove imidazole residues. Protein concentrations were determined using the Bradford assay (Bio-Rad, cat#500-0006) with a Bovine Serum Albumin (BSA) standard (FIG. 3).

1. additionFractions containing gamma-zein/YFP samples
2. additionAfter centrifugation, sterile PBS solution was added up to 15 ml for buffer exchange
3. customdialysis
4. customdialyzed against 2 L of PBS at 4° C. overnight
5. customto remove imidazole residues
6. concentrationProtein concentrations