Materials and Reagents

1. Desalting NAP5 columns (e.g., GE Healthcare, HiTrap Desalt, catalog number: 17-0853-01 )
   
2. Gel filtration columns
   We used prepacked GE 10/300 Tricorn columns, a Superdex 75 (GE Healthcare, catalog number: 17-5174-01 ) for small soluble proteins, or a Superose 6 for lipid-reconstituted samples (GE Healthcare, catalog number: 17-5172-01 ).
   
3. Proteins
   Notes:
   1. Metal binding proteins of interest must be on the hand in purified native state, with aggregates having been removed prior to the experiments.
      
   2. Proteins should be available in milligram quantities and should preferably have different molecular weights (which is a prerequisite for gel filtration).
      
   3. Proteins too small to be distinguished from each other on gel filtration columns could be fused to a polypeptide tag, e.g., the maltose binding protein or glutathione-S-transferase in order to artificially increase the molecular weight difference of the metal ion transfer pair.
      
   4. We used an eGFP tag because it is relatively inert in redox-reactions and facilitates easy detection and concentration determination.
      
   5. Care has to be taken with the use of a His-tag for means of protein purification. Although it was demonstrated that His-tags do not interfere with copper binding and quantification experiments under certain conditions (González-Guerrero et al., 2008), the metal chelating property of the hexahistidine sequence may cause metal carry-overs in some experiments.
4. 3-(N-morpholino) propanesulfonic acid (MOPS), buffer grade (Sigma-Aldrich, catalog number: M1254 )
   
5. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
   
6. Sodium ascorbate (Sigma-Aldrich, catalog number: 11140 )
   
7. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
   
8. Phosphatidylcholine (PC) (Sigma-Aldrich, catalog number: P3644 )
   
9. Sodium dodecylsulfate (SDS) (Sigma-Aldrich, catalog number: L6026 )
   
10. Bathocuproinedisulfonate (BCS) (Sigma-Aldrich, catalog number: B1125 )
    
11. Bicinchoninic acid (BCA) (Sigma-Aldrich, catalog number: D8284 )
    
12. β-dodecylmaltoside (β-DDM) (Sigma-Aldrich, catalog number: D5172 )
    
13. Copper sulfate (CuSO₄) (Sigma-Aldrich, catalog number: 451657 )
    
14. Buffer A (see Recipes)
    
15. BCA-copper buffer (see Recipes)
    
16. SDS solution (see Recipes)
    
17. Ascorbate solution (see Recipes)
    
18. BCS solution (see Recipes)
    

Equipment

1. (Optional) A dynamic light scattering instrument [e.g., High performance particle sizer (Malvern Instruments, model: HPP 5001 or Zetasizer Nano S )]
   
2. Ultrasonic homogenizer for preparation of lipid emulsions (e.g., BANDELIN electronic GmbH & Co, model: UP 400 S )
   
3. Ultrasonic bath for buffer degassing (e.g., BANDELIN electronic GmbH & Co, model: Sonorex DL510H )
   
4. An automated chromatography unit (e.g., GE Healthcare, Äkta Purifier or Explorer)
   Note: It is essential for the experiments. Use of a regular HPLC unit with fraction collector would be just as fine, provided that some modifications with respect to the tolerance of salt-containing elution buffers have been taken care of.
   
5. A thermostated shaking incubator for 1.5 ml type reaction vials (e.g., Eppendorf, catalog number: 5384000012 )
   
6. Centrifugal evaporator (e.g., Eppendorf, catalog number: 5305000304 )
   
7. Calibrated micropipette (e.g., Eppendorf, catalog number: 3120000062 ), alternatively: analytical balance (e.g., Mettler-Toledo International Inc., catalog number: 11142056 )
   
8. UV-VIS Spectrophotometer (e.g., Jasco, model: J-650 ) and Nanodrop spectrophotometer (Thermo Fisher Scientific), alternatively: UV-VIS spectrophotometer and small volume cuvette (e.g., Hellma, model: 105.250-QS or Eppendorf, µCuvette^®, model: G1.0 )
   
9. Quartz microcuvette (e.g., Hellma, catalog number: 104-10-40 104-QS )
   
10. Standard SDS-PAGE equipment (e.g., Bio-Rad Laboratories, catalog number: 1658001FC )
    

Procedure