Probing Yeast Protein Microarrays for Protein-protein Interactions Using V5-epitope Tagged Fusion Protein Probes

Materials and Reagents

1. Zirconia beads (Biospec Products, catalog number: 11079110zx 1.0mm dia)
   
2. His-tag affinity resin (His Spintrap, GE Life sciences, catalog number: 28-4013-53 )
   
3. Protein microarray (Life Technologies, catalog number: PAH0525101 )
   
4. V5-AlexaFluor 647 antibody (Life Technologies, catalog number: 451098 )
   
5. Glycerol (Sigma-Aldrich, catalog number: G5516 )
   
6. Potassium chloride
   
7. Sodium chloride
   
8. Triton X-100
   
9. Dithiothreitol (DTT) (Promega, catalog number: P1170 )
   
10. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-250MG )
    
11. Imidazole (Sigma-Aldrich, catalog number: I5513-5G )
    
12. ATP (for kinases)
    
13. BSA
    
14. Tween-20
    
15. Yeast extract
    
16. Peptone
    
17. Galactose
    
18. Yeast nitrogen base
    
19. (NH₄)₂SO₄
    
20. Raffinose (Sigma-Aldrich, catalog number: R0514-100G )
    
21. Agar
    
22. Lysis buffer (see Recipes)
    
23. Wash buffer 1 (see Recipes)
    
24. Wash buffer 2 (see Recipes)
    
25. Wash buffer 3 (see Recipes)
    
26. Elution buffer (see Recipes)
    
27. Probe buffer (see Recipes)
    
28. Blocking buffer (see Recipes)
    
29. 3x YEP-GAL (see Recipes)
    
30. Synthetic complete minus uracil media (Sc-ura) (see Recipes)
    

Equipment

1. FastPrep Cell Lyser (MP Bio, catalog number: 116004500 - 1 each )
   
2. High speed centrifuges
   
3. JA-10 (or comparable) rotor
   
4. Axon 4200 AL microarray reader
   
5. 50 ml conical tube
   
6. Wheel
   
7. Humidified chamber
   

Procedure