Mouse anti β actin igg

Non-detergent sodium carbonate solution was used to extract lipid microdomains (Zhang et al., 2005 (link)). HK-2 cells were grown to near confluence in 100-mm² dishes. After two washes with ice-cold phosphate-buffered saline, two confluent dishes were scraped into 1 ml of 500 mM sodium carbonate, pH 11.0, in the presence of 1 mM PMSF and protease inhibitors cocktail. Homogenization was carried out with 10 strokes using a tight-fitting Dounce homogenizer, followed by three 20-sec bursts at 30% of maximal power to disrupt cellular membranes. The homogenates were adjusted to 45% sucrose by addition of 1 ml of 90% sucrose prepared in MBS [2-(N-morpholino) ethanesulfonic acid-buffered saline, 25 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.5, and 0.15 M NaCl] and placed at the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient (6 ml of 35% sucrose and 4 ml of 5% sucrose, both prepared in MBS) was formed above and centrifuged at 39,000 rpm for 20 h in an SW40 TI rotor (Beckman Instruments). A light-scattering band was observed at the 3–35% sucrose interface. Twelve 1-ml fractions were collected from the top of the tubes, and equal portion of each fraction was separated by SDS-PAGE for Western blot analysis as described above. The primary antibodies were goat anti-hKIM-1, rabbit anti-clathrin, rabbit anti-caveolin-1, and mouse anti-β-actin IgG (Sigma–Aldrich).