Prepare sucrose gradients one day before use to allow gradients to become continuous. Make sterile15% and 50% sucrose solutions in polysome lysis buffer and store at 4°C . In order to pour the gradients, use 5ml pipettes. Add 5.5 mL of 15% sucrose to the bottom of an ultracentrifuge tube. Next add 5.5 mL of 50% sucrose solution under the 5% solution by placing the pipette tip near bottom of tube. We should observe clear separation of layers, indicating that minimal mixing has occurred. Store gradients at 4°C overnight in a flat horizontal position. Preparation of lysates from mammalian cells This protocol does not call for pretreatment of mammalian cells with cycloheximide. See: Ingolia et al. Nature Protocols Vol 7, No. 8, 2012 pg. 1534. Cell lysis: Aspirate medium from the 15 cm dish and place dish on ice. Rinse dish with 25 ml ice-cold PBS supplemented with 250 μL of cycloheximide. Aspirate the PBS and repeat wash. Make sure to remove all the PBS. Drip on the dish 800 μL of the polysome lysis buffer (no Superasin inhibitor if this lysate will be digested). Scrape the cells off the plate with a cell scraper and mix well with the lysis buffer, keeping the cells and the plate cold in ice. Mix the suspension several times with a pipette. Pass lysate five times through a 23G needle to homogenize. Clarify the lysate: centrifuge at max. speed at 4oC for 10 minutes. Transfer the supernatant to a new tube. Dilute the lysate 1:10 and measure the absorbance. For the polysome profiles on sucrose gradients usually I load at least 10 OD 260 units (i.e. 800 μL of a lysate with an absorbance at 260 of 1.3 when diluted ten-fold). Use the same amount of lysate for the pulldown. 10. Snap freeze the lysate in liquid nitrogen. Store at -80C. Centrifugation of gradients Thaw polyribosome lysates on ice.
Prepare sucrose gradients one day before use to allow gradients to become continuous. Make sterile15% and 50% sucrose solutions in polysome lysis buffer and store at 4°C . In order to pour the gradients, use 5ml pipettes. Add 5.5 mL of 15% sucrose to the bottom of an ultracentrifuge tube. Next add 5.5 mL of 50% sucrose solution under the 5% solution by placing the pipette tip near bottom of tube. We should observe clear separation of layers, indicating that minimal mixing has occurred. Store gradients at 4°C overnight in a flat horizontal position. Preparation of lysates from mammalian cells This protocol does not call for pretreatment of mammalian cells with cycloheximide. See: Ingolia et al. Nature Protocols Vol 7, No. 8, 2012 pg. 1534. Cell lysis: Aspirate medium from the 15 cm dish and place dish on ice. Rinse dish with 25 ml ice-cold PBS supplemented with 250 μL of cycloheximide. Aspirate the PBS and repeat wash. Make sure to remove all the PBS. Drip on the dish 800 μL of the polysome lysis buffer (no Superasin inhibitor if this lysate will be digested). Scrape the cells off the plate with a cell scraper and mix well with the lysis buffer, keeping the cells and the plate cold in ice. Mix the suspension several times with a pipette. Pass lysate five times through a 23G needle to homogenize. Clarify the lysate: centrifuge at max. speed at 4oC for 10 minutes. Transfer the supernatant to a new tube. Dilute the lysate 1:10 and measure the absorbance. For the polysome profiles on sucrose gradients usually I load at least 10 OD 260 units (i.e. 800 μL of a lysate with an absorbance at 260 of 1.3 when diluted ten-fold). Use the same amount of lysate for the pulldown. 10. Snap freeze the lysate in liquid nitrogen. Store at -80C. Centrifugation of gradients Thaw polyribosome lysates on ice.