Preparation of Nuclear and Cytosolic Fractions for CAR Sample Clauses

Preparation of Nuclear and Cytosolic Fractions for CAR blotting The following protocol was adapted from (Li, Lii, Yang, & Chen, 2007). After harvest cells were placed in a -20 °C freezer and 0.25 mL of cell lysis buffer (50 mM Tris, pH 7.5, 0.1 % SDS, 0.5 % Nonidet P-40, 1 mM EDTA, protease inhibitor mixture; Sigma-Xxxxxxx) was added to each well. Cells were collected with a cell scraper and the samples were briefly sonicated for five seconds at power 3. The samples were allowed to incubate on ice for 10 min while vortexing briefly and then centrifuged at 700g for five minutes at 4 °C. The supernatant was collected for the cytosolic fraction and the crude pellet was use for the nuclear fraction. To purify the nuclear fraction, 40 µL of buffer B (146 mM sucrose, 100 mM KCl, 5 mM MgCl2, 0.5 mM CaCl2, 10 mM Tris-HCL 7.5 pH, and 0.1 mM PMSF) was added to each sample. The samples were shaken for 30 minutes at 4 °C and then centrifuged at 1600 g for 10 min at 4 °C. The supernatant was removed and collected. The pellet was then rewashed with 50 µL of buffer B, shaken for 30 min at 4 °C, and centrifuged at 1600g for 10 min at 4 °C. The remaining supernatant was added to the previously collected supernatant and the total volume (90 µL) was used for the nuclear protein. For the cytosolic fraction, the samples were sonicated for seven seconds at power 15 and then centrifuged at 12000g for 10 min. The final supernatant was used for the cytosolic fraction and the amount of protein in each sample was analyzed using the BCA assay as previously described. Levels of CAR protein expression were observed using SDS-PAGE and western blotting as previously described.