RIBOSOMAL FRACTIONATION

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)