Procedure
RIBOSOMAL FRACTIONATION:
Run on a 25% sucrose cushion,
Pack a TLA 100.2 tube with 600ul of sucrose buffer, carefully layer 500 ul of lysate. Add 1ul of CHX (100mg/ml) to the cushion before applying the lysate
Spin in an ultracentrifuge at 75,000 RPM for 20 min @ 4°C
Take about 400ul of the lysate (carefully without disrupting the sucrose cushion) This is the (S) supernatant fraction (save 10ul for Total S fraction)
Remove sucrose buffer
Wash ribosomal pellet with 500ul of lysis buffer
Spin ribosomal resuspension at 10,000g for 10 min to remove debris
Remove supernatant, this is the (R) ribosomal fraction (save 10 ul for total R fraction)
Sucrose Buffer
20mM HEPES pH7.5
10mM MgCl2
50mM KCl
25% Sucrose w/v