Xcell surelock novex mini cell

Electrophoresis and protein transfer were performed using the XCell Surelock Novex Mini- Cell (Invitrogen, Carlsbad, CA) system. Protein lysates were separated by SDS-PAGE using pre-cast NuPAGE® Novex 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to a PVDF membrane (Millipore, Billerica, MA). Membranes were then incubated at room temperature for 1 h in blocking buffer supplemented with 5% BSA/PBS. After blocking, membranes were incubated overnight at 4°C with the following primary antibodies diluted 1:1000 in 5% BSA/PBS: STARS (Institute of Medical and Veterinary Science, Adelaide SA); Myosin, Sarcomeric (MF-20, Developmental Studies Hybridoma Bank, Iowa City IA); SRF (Santa Cruz Biotechnology, Dallas, TX (sc-335)). Following washing the membranes were incubated for 1 h with the corresponding infrared dye-conjugated secondary antibodies (Thermo Fisher Scientific, Inc, Rockford, IL). After washing, the specific proteins were revealed using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). GAPDH (G8795, Sigma-Aldrich, Castle Hill, NSW) was used to control for protein loading and individual protein band optical densities were determined using the Odyssey Infrared Imaging System software.

All sample preparations were conducted under clean conditions; cell samples (12 mL) were centrifuged (15 min, 30,000 g) and cell pellets were resuspended in PBS (10 mL) then centrifuged (2 min, 300 g) before resuspending in milliQ water (5 mL) and transferring to microcentrifuge tubes. To ensure complete removal of growth medium, resuspended cell pellets were centrifuged once more, supernatants discarded, and cell pellets resuspended in MilliQ water before a final centrifugation (18,000 g, 15 min). Supernatants were discarded and cell pellets were resolubilized in buffer containing 8 M urea, 100 mM DTT (dithiothreitol), 4% (w/v) CHAPS (3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate), 0.2% (v/v) carrier ampholytes (pH 3–10; Bio-Rad, Hercules, CA), 40 mM Tris Base (pH 7), and 0.02% (w/v) bromophenol blue. Samples were then vortexed and sonicated (30 s) before protein separation through 2DE (XCell SureLock Novex minicell, Invitrogen).