Immobilon western millipore chemiluminescence hrp substrate

Cells cultured on 100-mm tissue culture dishes were briefly rinsed with phosphate-buffered saline (PBS) and then scraped into a lysis buffer containing 50 mM Tris/HCl, pH 7.4, 10 mM MgCl₂, 150 mM NaCl, 1% Triton X-100, and EZBlock Protease Inhibitor Cocktail. The supernatant was collected after centrifugation at 14,000 rpm for 10 min. For immunoblotting, lysates were boiled in 2× Laemmli buffer, and 20 μg of protein was resolved by SDS–PAGE in each lane of a 13% gel. The proteins were transferred to polyvinylidene fluoride, immunoblotted with the indicated antibodies, and visualized using the Immobilon Western Millipore Chemiluminescence HRP substrate (Millipore). Blots were developed using either film or an Azure c600 chemiluminescence detector (Azure Biosystems).

Cells cultured on 100-mm tissue culture dishes were rinsed with TBS and 1 mM MgCl₂ and then scraped into a lysis buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% Triton X-100, and EZBlock protease inhibitor cocktail (BioVision). The supernatant was collected after centrifugation at 16,800 g for 10 min. For immunoblotting, lysates were boiled in 2 × Laemmli buffer, and were resolved by SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride and immunoblotted with the indicated antibodies. Immunocomplexes were visualized using the Immobilon Western Millipore Chemiluminescence HRP substrate (Millipore).