Okamoto et al. 10.1073/pnas.0701656104. |
SI Methods
Purification of CaMKII from Insect Cells.
Sf21 cells were plated at 1.2 ´ 108 cells per 500 cm2 triple surface plates. After 24 h, cells were infected with baculoviral particles containing CaMKII cDNA. To coexpress a and b subunits, the virus particles were titrated to give a ratio of CaMKIIa to b of »3:1, mimicking endogenous forebrain CaMKII. Three days after infection, cells were detached by gentle tapping on the sides and were pelleted at 1,500 ´ g for 5 min. Cells were gently washed with PBS and spun again; the pellet was stored at -70°C. The pellet was resuspended in 38 ml of buffer containing 10 mM Tris•HCl, 2.5% betaine, 1 mM EGTA, and 1 mM EDTA, pH 7.5, with a mixture of protease inhibitors (soybean trypsin inhibitor, leupeptin, PMSF) and sonicated. After ultracentrifugation, the supernatant was mixed slowly with an equal volume of saturated ammonium sulfate. The solution was centrifuged at 7,000 ´ g for 10 min, and the pellet was gently resuspended in 20 ml of CaM-Sepharose start buffer containing 40 mM Hepes, 100 mM NaCl, 10% glycerol, pH 7.4. Insoluble material was removed by another centrifuge spin at 7,000 ´ g for 10 min, and the supernatant was added to the CaM-Sepharose column equilibrated with CaM-Sepharose start buffer. The flow-through was reapplied to the column to maximally recover the kinase. The column was washed with 15 ml of start buffer and then with 10 ml of wash buffer (the same as start buffer except that it contains 1 M NaCl) and again with 10 ml of start buffer. CaMKII was eluted by using CaM-Sepharose elution buffer (the same as start buffer, except that CaCl2 was omitted and 2 mM EGTA and 0.5 M NaCl were added), and »1-ml fractions were collected. Fractions containing CaMKII as visualized from Coomassie Brilliant Blue staining of the gel were pooled and concentrated using Amicon Ultra 15 (Millipore).