Ribosome Fractionation in Yeast

Figure 1. Ribosome profiles after sucrose gradient centrifugation. Ribosome fractionation was performed in the presence of CHX (black), in the absence of CHX (red) or in the presence of EDTA (green). CHX stabilizes polysomes. In the absence of CHX polysomes are destroyed but 80S is still present. 80S can be completely disrupted in cell extracts by treatment with EDTA.

Materials and Reagents

1. Glass beads for cells breaking 0.5 mm (Bio Spec Products, catalog number: 110/9105 )
2. Cycloheximide (CHX) (Sigma-Aldrich, catalog number:  C7698 ) solution 100 mg/ml prepared on ethanol
3. Bradford reactive (Bio-Rad Laboratories, catalog number:  500-0006 )
4. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number:  13560400 )
5. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number:  P7625 ) 100 mM solution prepared on isopropanol
6. 100% trichloroacetic acid (TCA)  (Fluka, catalog number: 91230 )
7. Tubes for SW41 rotor Ti (Beckman Coulter, catalog number:  331372 )
8. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number:  1836170 )
9. Lysis buffer
10. Ethanol
    
11. Laemmli sample buffer
    
12. Bromophenol blue
    
13. YPD media (see Recipes)
    
14. Buffer A (see Recipes)
    
15. 2x buffer B2 times concentrated (see Recipes)
    
16. Sucrose gradient (see Recipes)
    
17. Foni-inert (see Recipes)
    

Equipment

1. Table Centrifuges
2. Glass bead beater Genie Disruptor (Scientific Industries, catalog number: SI-DD38 )
3. Ultracentrifuge
4. Rotor SW41 Ti (Beckman Coulter, catalog number:  331362 )
5. Density Gradient Fractionation System (ISCO, catalog number: 67-9000-177 )

Procedure