Preparation of Chloroplast Lipid Membrane and Lipid-protein Interaction Assay

Materials and Reagents

1. Preparation of lipids for chloroplast lipid membrane
   
   1. Amersham Protran Premium NC0.2 (GE Healthcare, Dharmacon, catalog number: AP-10600081 )
      
   2. TLC silica gel 60 plate (Merck Millipore Corporation, catalog number: 105721 )
   3. Fused silica capillary column, 0.25 mm x 50 m (Shinwa Chemical Industries, catalog number: HR-SS-10 )
      
   4. Cells of the cyanobacterium, Synechocystis sp. PCC 6803 (for lipid extraction)
      
   5. Methanol (Wako Pure Chemical Industries, Siyaku, catalog number: 137-01823 )
      
   6. Chloroform (Wako Pure Chemical Industries, Siyaku, catalog number: 038-02601 )
      
   7. 0.01% primuline (Sigma-Aldrich, catalog number: 206865 ), in 80% acetone
      
   8. Pentadecanoic acid (Sigma-Aldrich, catalog number: 91446-5G )
      
   9. Methanolic HCl (Sigma-Aldrich, catalog number: 33050-U ), dilute with methanol 3 times
      
   10. Hexane (Wako Pure Chemical Industries, Siyaku, catalog number: 085-00411 )
   
   
2. Lipid-Protein interaction assay
   
   1. PIP Strips, Membrane Lipid Strips (Echelon Biosciences, catalog numbers: P-6001 and P-6002 )
      
   2. Hybri-Bag (Cosmo Bio, catalog number: S-1001 )
      
   3. Albumin from bovine serum (BSA), fatty acid free (Wako Pure Chemical Industries, Siyaku, catalog number: 017-15146 )
      
   4. Purified tagged proteins
      
   5. An anti-penta-His mouse monoclonal antibody (QIAGEN, catalog number: 34660 ) and an anti-GST mouse monoclonal antibody (Sigma-Aldrich, catalog number: G1160 )
      
   6. Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, catalog number: 31432 )
      
   7. HRP substrate solution (Thermo Fisher Scientific, Pierce, catalog number: NCI32132 )
      
   8. Phosphate (pH 7.4)
      
   9. NaCl
      
   10. 0.1 % Tween-20
       
   11. Phosphate buffered saline (PBS) (see Recipes)
       
   12. PBST (see Recipes)

Equipment

1. Glass tubes (Iwaki, catalog numbers: 71-088-004 and 71-063-006 )
   
2. Pasteur pipette (Sansyo, Iwaki, catalog number: IK-PAS-9P )
   
3. Spectrophotometer (Scinteck Instruments, Pharmacia Biotech, model: Ultrospec 2000 )
   
4. Vortex mixer
   
5. Centrifuge (TOMY SEIKO CO., model: LC-120 )
   
6. Vacuum dryer (Shimadzu Corporation, model: SPE-200 centrifuge evaporator )
   
7. TLC developing chamber (AS ONE, catalog number: CK-0544-060 )
   
8. Hair dryer (commercially available)
   
9. UV illuminator
   
10. Razor blade
    
11. Heat block
    
12. Gas chromatograph (Shimadzu Corporation, model: GC-18A )
    
13. Fused silica capillary column, 0.25 mm x 50 m (Shinwa Chemical Industries, catalog number: HR-SS-10 )
    
14. Data processor (Shimadzu Corporation, model: C-R2AX )
    

Procedure