Materials and Reagents

1. W303a wild type yeast cells
   
2. Tris base (C₄H₁₁NO₃) (Thermo Fisher Scientific, catalog number: 77-86-1 )
   
3. NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
   
4. Na₂EDTA•2H₂O (EDTA) (Sigma-Aldrich, catalog number: ED2SS )
   
5. Triton X-100 (Thermo Fisher Scientific, catalog number: 9002-93-1 )
   
6. Phenylmethanesulfonyl fluoride (PMSF) (C₇H₇FO₂S) (Sigma-Aldrich, catalog number: P7626 )
   
7. Complete protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
   
8. PhosSTOP tablet (F. Hoffmann-La Roche, catalog number: 04906837001 )
   
9. Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
   
10. MgCl₂ (USB, catalog number: 18641 500 GM )
    
11. Dithiothreitol (DTT) (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 20290 )
    
12. Rapamycin (Santa Cruz Biotechnology, catalog number: sc-3504 )
    
13. Glass beads (Sigma-Aldrich, catalog number: G-8772 )
    
14. HA antibody (12CA5) (Abcam, catalog number: ab16918
    
15. ATP (Sigma-Aldrich, catalog number: A2383-1G )
    
16.  [γ-³²P]-ATP (PerkinElmer, catalog number: BLU002250UC )
    
17. Coomassie Blue R250 (National Diagnostics, catalog number: HS-605 )
    
18. HCl
    
19. 2.5x SDS loading dye
    
20. IP buffer (see Recipes)
    
21. Kinase buffer (see Recipes)
    
22. PBS buffer (see Recipes)
    

Equipment

1. Standard bench-top centrifuge
   
2. Shaker
   
3. 1.5 ml Eppendorf tubes
   
4. Authoradiograph
   

Procedure