0.45 um nitrocellulose membrane

Cells in culture were collected via trypsinization and pelleted via centrifugation. Cell pellets were lysed in AZ lysis buffer (50mM Tris pH 8, 250mM NaCl, 1% NP-40, 0.1% SDS, 5mM EDTA, 10mM Na4P2O7, 10mM NaF, 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), 1x PhosSTOP (Roche)). The protein concentration of each sample was determined using the DC™ (detergent compatible) protein assay (Bio-Rad Laboratories, Inc.). Protein concentrations were normalized and samples were prepared with 5x Laemmli sample buffer. Samples were run on a gradient gel (Bio-Rad) and transferred for Western blot on 0.45um Nitrocellulose membrane (Bio-Rad).
The primary antibodies used were mouse anti-PTEN (sc7974, Santa Cruz Biotechnology), and mouse anti-Beta Actin (Cell Signaling Technology #4970). Primary antibodies were used at 1:1000 dilutions and were incubated for 1 to 2 hours at room temperature or overnight at 4°C. Secondary goat anti-mouse antibody (Thermo Fisher Scientific/Pierce) or were used at a 1:10,000 dilution for 1 hour at room temperature. Primary and secondary antibodies were prepared in 5% milk. Three washes with Tris Buffered Saline with Tween 20 were each performed after primary incubation and after secondary incubation. Membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).