Materials and Reagents

1. BL21 Escherichia coli (E. coli) cells containing a plasmid for expression of the recombinant GST-MTV1 fusion protein (the bait)
   Note: In this exemplary case, the MTV1 coding sequence was cloned into a modified pGEX-2T plasmid (General Electric Company, catalog number: 28-9546-53 ), in which the multiple cloning site had been replaced by a Gateway cloning cassette (Note 1).
   
2. BL21 E. coli cells expressing the GST tag alone as negative control
   
3. Murashige and Skoog medium mix with vitamins and MES buffer (Duchefa Biochemie BV, catalog number: M0255.0010 )
   
4. Glutathione agarose (Sigma-Aldrich, catalog number: G4510 )
   
5. Complete inhibitor (EDTA free) (Roche Diagnostics, catalog number: 11 873 580 001 )
   
6. Phenylmethylsulfonyl fluoride PMSF (e.g. Sigma-Aldrich, catalog number: P7626 )
   
7. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
   
8. Carbenicillin (e.g. Sigma-Aldrich, catalog number: C9231 ) (Note 2)
   
9. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (e.g. Sigma-Aldrich, catalog number: I6758 )
   Note: Should be prepared as aqueous stock solution of 1 M and stored at -20 °C.
   
10. Sodium dodecyl sulfate (SDS) (e.g. Sigma-Aldrich, catalog number: L3771 , or any other supplier)
    Note: An aqueous stock solution of 20% (weight/volume) can be prepared and sterilized by autoclaving at 121 °C for 15 min.
    
11. Glycerol (e.g. Sigma-Aldrich, catalog number: G5516 , or any other supplier)
    
12. Tris (hydroxymethyl) aminomethane (Tris) (e.g. Sigma-Aldrich, catalog number: 252859 , or any other supplier)
    
13. Yeast extract (e.g. Sigma-Aldrich, catalog number: Y1625 , or any other supplier)
    
14. Tryptone (e.g. Sigma-Aldrich, catalog number: 95039 , or any other supplier)
    
15. Anti-GST polyclonal antibody (optional) (Carl Roth, catalog number: 3998 )
    
16. Anti-CHC monoclonal antibody (optional) (BD Biosciences, catalog number: 610499 )
    
17. Liquid nitrogen
    
18. NaCl (any supplier)
    
19. KCl (any supplier)
    
20. Na₂HPO₄ (any supplier)
    
21. KH₂PO₄ (any supplier)
    
22. β-mercaptoethanol
    
23. Liquid grown Arabidopsis seedlings of 6-8 days (see Recipes)
    
24. MS medium (see Recipes)
    
25. PBS (see Recipes)
    
26. Wash buffer (see Recipes)
    
27. Extraction buffer (see Recipes)
    
28. Sample loading buffer (see Recipes)
    
29. Liquid Lysogeny Broth (LB) growth medium (see Recipes)
    

Equipment

1. Microcentrifuge for 1.5 and 2 ml standard reaction tubes (any manufacturer)
   Note: Either refrigerated or situated in a 4 °C cold-room, should be able to reach 16,000 x g.
   
2. Refrigerated centrifuge for 50 ml conical “Falcon” type tubes (any manufacturer)
   Note: Should reach 4 °C and 3,000 x g.
   
3. Sonicator device (micro tip sonotrode type)
   Note: We use the labsonic model of B. Braun, which is, however, no longer produced. But any tip style sonicator device that is suitable for small volumes (2-5 ml) will work, for example the UP100H device coupled to the MS3 sonotrode (Hielscher Ultrasound Technology).
   
4. Poly-Prep Chromatography columns (Bio-Rad Laboratories, catalog number: 731-1550 )
   
5. Erlenmeyer flasks
   
6. Paper towels
   
7. 1.5 ml microcentrifuge tubes capable of supporting 16,000 x g (any supplier)
   
8. 50 ml polypropylene conical centrifuge tubes “Falcon” type (any supplier)
   
9. 0.20 µm filter unit (e.g. Minisart^®, Sartorious, catalog number: 17597 ) plus compatible 5 ml syringe
   
10. Shaking incubator for bacteria 37 °C (any manufacturer)
    
11. Shaking incubator 25 °C or orbital shaker at room temperature (any manufacturer)
    
12. End-over-end (orbital) mixer (any manufacturer)
    
13. Spectrophotometer capable of measuring optical density at 600 nm (any manufacturer)
    
14. Mortar and pestle (about 10 cm diameter) (any manufacturer)
    

Procedure