Image station 440mm pro

Cells were detached, centrifuged, extensively washed with PBS and lysed by the addition of ice-cold buffer containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100 (v/v), protease inhibitor cocktail Complete (Roche) and phosphatase inhibitor Cocktail 2 and 3 (Sigma). After 20 min incubation on ice, the lysate was centrifuged 10 min at 10,000 × g at 4 °C. The supernatant was collected and protein concentration was determined by the Bradford method. Equal amounts of protein were loaded on 12% SDS-PAGE, blotted on Immobilon-P membranes (Millipore), processed by western blot with the indicated antibody, and detected by chemiluminescence on a Kodak Image Station 440MM PRO. Quantitation of the signal was obtained by analysis with the Kodak 1D Image software.

Equal amounts of cellular proteins (10 μg) were loaded on 11% SDS/PAGE, blotted on Immobilon-P membranes (Millipore), processed by western blot with the indicated antibodies, and detected by chemiluminescence on a Kodak Image Station 440MM PRO. Quantitation of the signal was obtained by analysis with the Kodak 1D Image software.
Akt immunoprecipitations were performed from 50 μg of total protein lysates, from HA-tagged Akt isoforms transfected cells, in 30 μl of final volume. Lysates were incubated overnight at 4°C with 1.3 μl of anti-HA antibody followed by 25 μl protein A-Sepharose (Sigma-Aldrich) for 40 min at 4°C. Immunocomplexes were washed twice with Tris/HCl 50 mM (pH 7.5), once with Tris/HCl 5 mM (pH 7.5) and used as CK2 substrate in in vitro phosphorylation assays.

For lysate preparation, cells were lysed as described in [15 (link)]. Protein concentration was determined by the Bradford method. Equal amounts of proteins were loaded on 11% SDS-PAGE, blotted on Immobilon-P membranes (Millipore), and processed in Western blot (WB) with the indicated antibody, detected by chemiluminescence. Quantitation of the signal was obtained by chemiluminescence detection on a Kodak Image Station 440MM PRO and analysis with the Kodak 1D Image software.