×
Please leave your comments or questions
 

RIBOSOMAL FRACTIONATION

Author: Stefanie Duttler, updated date: , view: 12, Q&A: 0
Tags: Fractionation and Ribosome

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





Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.