Materials and Reagents

1. Cell crude extract
   
2. Purified protein or crude extract of ubiquitin activating enzyme (E1)
   
3. Purified recombinant ubiquitin conjugating enzyme (E2) fused with a protein tag (such as 6x His tag)
   
4. Purified recombinant ubiquitin ligase (E3)
   
5. Purified ubiquitin or recombinant ubiquitin protein fused with a tag ( (Kraft et al., 2005; Liu et al., 2010 )
   
6. ATP (Sigma-Aldrich, catalog number: A7699 )
   
7. MgCl₂ (Sigma-Aldrich, catalog number: V900020 )
   
8. Anti-ubiquitin antibody or antibody of a certain protein tag
   For example:
   Anti-Ub, raised in our laboratory
   Anti-His (Santa Cruz, catalog number: sc-0836 )
   Nickel-HRP (KPL, Kirkegaard & Perry Laboratories, catalog number: 24-01-01 )
   Anti-GST (Beijing Protein Innovation, catalog number: AbM59001-2H5-PU )
   Anti-MBP (New England Biolabs, catalog number: E8030S )
   
9. Amylose resin (New England Biolabs, catalog number: E8021 )
   
10. DTT
    
11. SDS-PAGE gel
    
12. Tris
    
13. PMSF
    
14. Skimmed milk powder or BSA
    
15. Chemiluminescent HRP substrate kit (EMD Millipore, catalog number: WBKLS0100 )
    
16. Glycerol
    
17. Bromophenol blue
    
18. MBP (maltose binding protein) Column buffer (see Recipes)
    
19. 20x reaction buffer (see Recipes)
    
20. 4x SDS sample buffer with or without DTT (or β- mercaptoethanol) (see Recipes)
    
21. 1x PBS (see Recipes)
    

Equipment

1. Centrifuge
   
2. Thermo-mixturer (Eppendorf Thermomixer comfort)
   
3. Protein electrophoresis apparatus
   
4. Western blot apparatus
   

Procedure