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Author: Stefanie Duttler, updated date: , view: 311, Q&A: 0
Tags: Fractionation and Ribosome



  • 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

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