Protein Fractionation from Brain Cortex

Sequential fractionation using RAB, RIPA, 70% formic acid (FA) buffer was performed as described previously^{36 (link)} with modifications. Briefly, cortices from left hemisphere were weighted and homogenized using a dounce homogenizer (DWK Life Science #357422) for 25 strokes in 10-fold volume of ice-cold RAB buffer (100 mM MES, 1 mM EGTA, 0.5 mM MgSO₄, 750 mM NaCl, 20 mM NaF, 1 mM Na₃VO₄, pH 7.0, supplemented with PhosSTOP, cOmplete-mini (Roche)). After ultracentrifugation for 20 min at 50,000 × g at 4 °C, the supernatant was collected and saved as the RAB-soluble fraction and the pellet was resuspended in 10-fold volume of ice-cold RIRA buffer (25 mM Tris, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 20 mM NaF, 1 mM Na₃VO₄, pH 8.0, supplemented with PhosSTOP, cOmplete-mini (Roche)) and nutated for 30 min at 4 °C. After ultracentrifugation for 20 min at 50,000 × g at 4 °C, the supernatant was collected and saved as the RIPA-soluble fraction and the pellet was resuspended in 3-fold volume of ice-cold 70% FA and nutated for 30 min at 4 °C. After ultracentrifugation for 20 min at 50,000 × g at 4 °C, the supernatant was collected and saved as the FA-soluble fraction. All fractions were stored at –80 °C.