Nupage bistris 4 12 gradient gel

Lysates for SDS-PAGE analysis were prepared in lithium dodecylsulfate sample buffer (Life Technologies) and 25 mM TCEP. Samples were heated to 65 °C for 5 min and then loaded onto a NuPage BisTris 4–12% gradient gel (Life Technologies), in either MOPS, or MES buffer. Proteins were electrophoresed and then wet-transferred to nitrocellulose membranes at 35 V for 1.5–2 h. Membranes were then blocked in 5% BSA in immunoblot wash buffer (TBS + 0.1% Tween-20) for 1 h at room temperature. Membranes were then probed with primary antibody overnight at 4°C, washed and then re-probed with IRdye-conjugated secondary antibodies. All antibodies are listed in
Table 1.

Lysates for SDS-PAGE analysis were prepared in lithium dodecylsulphate sample buffer (Life Technologies) and 25 mM TCEP. Samples were heated to 65°C for 5 min and then loaded onto a NuPage BisTris 4–12% gradient gel (Life Technologies), in either MOPS, or MES buffer. Proteins were electrophoresed and then wet transferred to nitrocellulose membranes at 35 V for 1.5 hr. Membranes were then blocked in 5% BSA in immunoblot wash buffer (TBS +0.1% Tween-20) for 1 hr at room temperature. Membranes were then probed with primary antibody overnight at 4°C, washed and then re-probed with LiCor dye-conjugated secondary antibodies (either IRDye-688 or IRDye-800). Primary antibodies for cell cycle immunoblot analysis were obtained from Cell Signaling Technology (cyclin B1, cyclin A, cyclin E, CDT1). Bands were visualised using the Odyssey CLx scanner (LiCor Biosciences).