Xcell surelock mini gel tank

All 2D samples that were not spiked with recombinant proteins were run on 12-well 1 mm thick 4-12% NuPAGE Bis-Tris gels (Cat. No. NP0322, Thermo Fisher Scientific). Each sample loaded consisted of lysate diluted in 2D lysis buffer and the appropriate amount of NuPAGE LDS Sample Buffer (Cat. No. NP0008, Thermo Fisher Scientific), and NuPAGE Sample Reducing Agent (Cat. No. NP0009, Thermo Fisher Scientific). Protein separation was performed immediately following gel loading at 200 V, 120 mA, and 25 W for 55 min in NuPAGE MOPS SDS Running Buffer (Cat. No. NP0001-02, Thermo Fisher Scientific). Proteins were then transferred onto Optitran BA-S 85 Reinforced Nitrocellulose (Cat. No. 10439196, GE Life Sciences) or Immobilon-PSQ Polyvinylidene Difluoride (PVDF) membrane (Cat. No. ISEQ00010, EMD Millipore) in 1× NuPAGE Transfer Buffer (Cat. No. NP0006-1, Thermo Fisher Scientific) containing 10% methanol. PVDF membranes were prewet in 100% methanol for 1 min before transfer. Proteins were transferred at 25 V, 120 mA, and 17 W for 1 h and 10 min. SDS-PAGE and transfer steps were performed in an XCell SureLock Mini Gel Tank (Thermo Fisher Scientific) on a Power Zoom-dual power pack or Power Ease 500 power pack (Thermo Fisher Scientific).