Materials and Reagents

1. Nicotiana benthamiana seeds
   
2. Agrobacterium strains harboring binary vector (e.g. pCambia1300, pBIN19) with the targeted gene fused with protein purification tag
   Note: In this protocol, we use 3x FLAG tag as an example.
   
3. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
   
4. MES (pH 5.6) (Sigma-Aldrich, catalog number: M8250 )
   
5. Protease Inhibitor (Roche, catalog number: 04693132001 )
   
6. Nonidet P 40 substitute (Fluka, catalog number: 74385 )
   
7. Induction medium (see Recipes)
   
8. Infiltration solution (see Recipes)
   
9. Extraction buffer (see Recipes)
   
10. TBS (see Recipes)
    
11. 4x SDS (see Recipes)
    

Equipment

1. Anti-FLAG beads (Sigma-Aldrich, catalog number: A2220 )
   
2. 3x FLAG peptides (Sigma-Aldrich, catalog number: F3290 )
   
3. Protein G sepharose beads (GE healthcare, catalog number: 17-0618-01 )
   
4. Test tubes, flask and shaker machine for culturing Agrobacterium
   
5. Centrifuge
   
6. 1 ml blunt syringe (BD, catalog number: 309659 )
   

Procedure