Mini protean tetra vertical electrophoresis system

The proteomic responses of B2 oysters to ocean acidification and warming were assessed by two-dimensional gel electrophoresis (2DE). A total of three gels were run per treatment (representing each of the three replicate tanks per treatment). Proteins (150 μg in 125 μl of rehydration buffer containing 0.2% pharmalytes) were immobilised in pH linear gradient gel strips (7 cm, pH 4–7; ReadyStrip™ IPG Strips, Bio-Rad) by overnight passive rehydration. Isoelectric focusing (IEF) was performed using an IPGphor IEF System (GE Healthcare) at 100 V for 2 h, 250 V for 20 min, a gradient up to 5000 V for 2 h and then 5000 V for 2 h. Following IEF, gel strips were equilibrated for 20 min in equilibration buffer I (1% DTT, 75 mM of 1.5 M Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) and then for 20 min in equilibration buffer II (2.5% iodoacetamide instead of 1% DTT in equilibration buffer I). Second dimension separation was conducted using 12% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad) in a Mini-PROTEAN® Tetra Vertical Electrophoresis System (Bio-Rad). After electrophoresis, gels were stained with blue silver [41 (link)] and visualised using a ChemiDoc XRS+ (Bio-Rad). Quantitative image analysis of protein spots was performed by PDQuest 2-D Analysis Software (Bio-Rad).

The Mini-PROTEAN Tetra Vertical Electrophoresis System (Bio-Rad, cat. # 1658000FC) was used to hand cast polyacrylamide gels. Forty percent acrylamide/bis solution (37.5:1, 2.6% crosslinker) and other reagents for gel casting and running were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Protein samples were denatured with Laemmli buffer [1 (link)] and applied to 8% polyacrylamide gels for electrophoresis.
Following electrophoresis, the gel was placed in a solution of 40% methanol and 10% acetic acid containing 0.25% Coomassie Brilliant Blue R-250 (Fisher Scientific, Inc., cat. # BP101, Fair Lawn, NJ) for 2 h. The gel was de-stained with several changes of distilled water overnight until the background was transparent. A stain-free method was also used to visualize proteins. 2,2,2-trichloroethanol (TCE) (Sigma, St. Louis, MO) was added to the separating gels at a concentration of 0.5%. Proteins were visualized under ultraviolet (UV) light for 5 min [2 (link)].

The level of α-amylase protein in media was qualitatively analyzed using an α-amylase-specific aptamer labeled with fluorescence. The sequence of A. oryzae α-amylase aptamer used in this study (Table 1) was designed following that described in a previous study [33 (link)].
Following RF-EMF exposure, the spore suspension was incubated at 30 °C and shaken for 16 h. The cultured media was collected, and 5 μL of media was mixed with or without 3 μL of 10 μM α-amylase aptamer labeled with fluorescence. The mixture was incubated at 25 °C in the dark for 3 h. The commercially available solution of A. oryzae α-amylase (1 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. After incubation, the mixture solution was applied to native polyacrylamide gel (8% in 1 × TBE) electrophoresis. Electrophoresis was processed at output voltage 80 V for 40 min using a Mini-PROTEAN^® Tetra Vertical Electrophoresis System (BioRad, Hercules, CA, USA). After electrophoresis, the gel was examined and photographed using the ChemiDocTM MP Imaging System (BioRad, Hercules, CA, USA). The α-amylase band was detected at 532 nm and the intensity of the band was analyzed using the Image Lab Touch Software version 3.0.1 (BioRad, Hercules, CA, USA).