Materials and Reagents

1. 15 ml centrifuge tubes with conical bottoms
   
2. 50 ml centrifuge tubes with conical bottoms
   
3. 500 ml centrifuge tubes with conical bottoms
   
4. 1.5 ml microcentrifuge tubes
   
5. 1 ml syringe
   
6. 1 ml Bio-Scale mini Macro-Prep high Q ion exchange column (Bio-Rad Laboratories, catalog number: 7324120 )
   
7. Amicon Ultra-4 centrifugal filter (30,000 NMWL) (EMD Millipore, catalog number: UFC803024 )
   
8. Amicon Ultra-100 centrifugal filter (100,000 NWML) (EMD Millipore, catalog number: UFC910024 )
   
9. Diatoms
   
10. Liquid nitrogen
    
11. Polyvinylpolypyrrolidone (PVPP; insoluble) (Sigma-Aldrich, catalog number: 77627 )
    
12. Additional materials To test for Rubisco activity (Optional):
    Labelled CO₂ (as NaH¹⁴CO₃) (5 mCi) (PerkinElmer, catalog number: NEC086H005MC )
    Ribulose-1,5-bisphosphate (RuBP; synthesized, purified and stored anaerobically as described in Kane et al., 1998)
    
13. Acetic acid (Sigma-Aldrich, catalog number: A9967 or 27225 )
    Note: The product acetic acid ( A9967 ) has been discontinued.
    
14. Methanol (Sigma-Aldrich, catalog number: M1770 or 494437 )
    Note: The product Methanol ( M1770 ) has been discontinued.
    
15. Soluble protein (as determined by Bradford assay) (Coomassie Plus Assay Kit) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23236 )
    
16. Additional materials to test for protein using Native and SDS-PAGE (Optional):
    Gel code blue stain (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 24590 )
    4-12% Tris-glycine mini gels (Thermo Fisher Scientific, Invitrogen^TM, catalog number: XV04120PK20 )
    4-12% Bis-Tris gels (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NP0321PK2 )
    SDS reducing buffer for SDS-PAGE (see Recipes)
    TBS buffer for SDS-PAGE (see Recipes)
    AttoPhos reagent (Astral Scientific, Gymea, NSW, Australia)
    Antisera raised against the large subunit holoenzyme of Rubisco in Phaeodactylum tricornutum
    Alkaline Phosphatase conjugated secondary antibody
    
17. 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) (Sigma-Aldrich, catalog number: E9502 )
    
18. Ethylenediaminetetraacetic acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E5134 )
    
19. Dithiothreitol (Sigma-Aldrich, catalog number: D0632 )
    
20. Plant protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
    
21. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    
22. Triethanolamine (Sigma-Aldrich, catalog number: 90279 )
    
23. Magnesium acetate (Sigma-Aldrich, catalog number: M5661 )
    
24. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    
25. Extraction buffer (see Recipes)
    
26. Column buffer (see Recipes)
    
27. Column elution buffer (see Recipes)
    
28. Specificity (S_C/O) buffer (see Recipes)
    

Note: All chemicals are of A.C.S. grade.

Equipment

1. French pressure cell press (Thermo Fisher Scientific, model: FA-078 )
   
2. Coulter Counter Z Series (Beckman Coulter, model: Z Series Coulter Counter )
   
3. Centrifuge (large volumes [1 L] at 2,000 x g, small volumes [< 15 ml] at 17, 600 x g, 4 °C)
   
4. Fume hood
   
5. Superdex 200 (GE Healthcare, catalog number: 17517501 )
   
6. FPLC (Äkta Pure 25) setup at 4 °C for size-exclusion chromatography using Superdex 200/30 (GE Healthcare, model: Äkta Pure 25 )
   
7. To confirm purification and activity of Rubisco:
   
1. Protein transfer apparatus and immunoblot imagining equipment – to check abundance and purity of extracted Rubisco
   
2. Radioisotope laboratory and associated septum capped vials and syringes

Procedure