Bead ruptor 24 elite homogenizer

Perigonadal mouse tissue (40–50 mg) was prepared in RIPA buffer (Sigma-Aldrich, R0278, St. Louis, MO) at a 1:3 ratio relative to tissue weight in 0.5 ml bead mill tubes with 1.4 mm ceramic beads (Omni International, 19626, Keenesaw, GA). All buffers contained protease inhibitors (Sigma, P-8340, St. Louis, MO) and phosphatase inhibitor cocktail set II (Millipore, 524625, Burlington, MA) and III (Millipore, 524627, Burlington, MA) at a 1:100 dilution. Samples were homogenized at 4°C using the Bead Ruptor 24 Elite Homogenizer (Omni International, 19–040E, Keenesaw, GA) at the following settings: Power: 5 m/s, Time: 30 sec, 1 cycle. The contents were spun down at max speed at 4°C for 10 min, resulting in three layers: a fat layer (top), the supernatant (middle), and the pellet (bottom). The supernatant was removed and was spun down again at max speed at 4°C for 10 min. The second supernatant was extracted and solubilized in 5X Laemmli buffer (50% glycerol, 500 mM DTT, 7.5% SDS, 300 mM Tris base, pH 6.8, 0.1% bromophenol blue) to a 1X dilution [25 (link)]. Protein concentrations were obtained using the Pierce 660 nM Protein Reagent (Thermo Fisher Scientific, 22660, Rockford, IL) with the addition of the IDCR reagent (Thermo Fisher Scientific, 22663, Rockford, IL). Samples were heated for 10 min at 95°C, then stored at −80°C.

Mouse ventricles (10–15mg) were prepared in standard relaxing buffer (SRB: 75 mM KCl, 10 mM Imidiazole pH 7.2, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3) at a 1:10 ratio relative to tissue weight. All buffers contained protease inhibitors (Sigma, P-8340, St. Louis, MO) and phosphatase inhibitor cocktail (Millipore, 524624, Burlington, MA) at a 1:100 dilutions as well as 100nM Calyculin A (Cell Signaling Technology, 9902, Danvers, MA) solubilized in DMSO. Samples were homogenized at 4°C using the Bead Ruptor 24 Elite Homogenizer (Omni International, 19–040E, Keenesaw, GA) at the following settings: Power: 5 m/s, Time: 15 sec, 3 cycles, 3 minute dwell between each cycle. Homogenized samples were re-suspended with Industrial Sample Buffer (ISB: 8 M urea, 2 M thiourea, 50 mM Tris pH 6.8, 3% SDS, 75 mM DTT, 0.05% bromophenol blue) [24 (link)] or 2X Laemmli Sample Buffer (Bio-Rad, Inc., cat#1610737, Hercules, CA) at 1:5 ratio relative to the homogenized protein and vortexed for 15 min. The samples were sonicated in a water bath for 10 min and underwent one freeze/thaw cycle. The samples were heated for 3 min at 100°C then spin clarified at room temperature at 21,000 x G for 3 min. Concentrations were obtained using the Pierce 660 nM Protein Assay with the addition of the IDCR reagent (Thermo Fisher Scientific, 22660, Rockford, IL). Samples were stored at −80°C.