Immobilin p pvdf membrane

Whole cell lysates were prepared from frozen cell pellets or flash frozen tumor samples using RIPA lysis buffer supplemented with 1× HALT protease and phosphatase inhibitors (Pierce, Thermo Fisher). Cell pellets were resuspended in 5 volumes of cold lysis buffer and incubated on ice for 15 minutes followed by sonication for 10 s with a 200V microtip sonicator set to 40% amplitude. Lysates were quantified and normalized using a BCA protein assay kit (Pierce, Thermo Fisher). Samples were denatured at 75C for 10 minutes in NuPAGE LDS sample buffer with NuPAGE sample reducing agent. 15uL of total protein was loaded and resolved on a precast 4%–12% Bis-Tris Gel (NuPAGE, Invitrogen). Gels were wet-transferred to a 0.45μM Immobilin-P PVDF membrane (Millipore) for chemiluminescent detection. Films were incubated at room temperature in TBS (Fisher) supplemented with 0.1% Tween20 (Fisher) and 5% non-fat dry milk for blocking. Blots were then incubated overnight at 4C with primary antibody diluted in TBS (Fisher) supplemented with 0.1% Tween20 (TBS-T) (Fisher) and 5% BSA (Cell Signaling). Blots were then washed with TBS-T 3× and incubated at room temperature for 1 hour with the relevant secondary antibody diluted in TBS-T and 5% non-fat dry milk. Blots were detected using ECL Western Blotting Substrate (Pierce).