Exercise 8.1 - Isolation of Chloroplasts from Spinach Leaves

LEVEL I

Materials

• Fresh spinach leaves
  
• Grinding solution
  
  • 0.33 M Sorbitol
    
  • 10 mM Sodium pyrophosphate (NaPO)
    
  • 4 mM MgCl
    
  • 2 mM Ascorbic Acid
    
  • Adjust pH to 6.5 with HCl
    
• Chopping board and knife
  
• Chilled mortar and pestle
  
• Cheesecloth
  
• Refrigerated preparative centrifuge
  
• Suspension solution
  
  • 0.33 M Sorbitol
    
  • 2 mM EDTA
    
  • 1 mM MgCl
    
  • 50 mM HEPES
    
  • Adjust pH to 7.6 with NaOH
    
• Hemacytometer and microscope

Procedure

1. Prepare an ice bath and pre-cool all glassware to be used.

2. Select several fresh spinach leaves and remove the large veins by tearing them loose from the leaves. Weigh out 4.0 grams of deveined leaf tissue.

3. Chop the tissue as fine as possible. Add the tissue to an ice-cold mortar containing 15 ml of grinding solution and grind to a fine paste.

4. Filter the solution through double layered cheesecloth into a beaker and squeeze the tissue pulp to recover all of the suspension.

5. Transfer the green suspension to a cold 50 ml. centrifuge tube and centrifuge at 200 xg for 1 minute at 4° C to pellet the unbroken cells and fragments.

6. Decant the supernatant into a clean centrifuge tube and recentrifuge at 1000 xg for 7 minutes. The pellet formed during this centrifugation contains chloroplasts. Decant and discard the supernatant.

7. Resuspend the chloroplast pellet in 5.0 ml. of cold suspension solution or 0.035 M NaCl. Use a glass stirring rod to gently disrupt the packed pellet. This is the chloroplast suspension for use in subsequent procedures.

8. Enclose the tube in aluminum foil and place it in an ice bucket. 1

9. Determine the number of chloroplasts/ml of suspension media using a hemocytometer.

Notes

The isolation procedure used here leaves the chloroplast outer membrane intact.If you wish to study the enzymes for photophosphorylation, wash the chloroplasts and rupture the outer membranes. To rupture the outer membranes, resuspend the chloroplasts in diluted suspension solution (1:25). Immediately centrifuge the chloroplast suspension at 8,000 xg for 5 minutes to collect the chloroplasts. Remove the diluted suspension media and resuspend the chloroplasts in isotonic media (0.35 M NaCl or undiluted suspension buffer).

Return to Table of Contents