Isolation of Chloroplasts from Spinach Leaves

Procedure

1. Prepare an ice bath and precool all glassware to be used (including a mortar and pestle).
2. Select several fresh spinach leaves. Remove the large veins by tearing them loose from the leaves. Weigh out 4 g of deveined leaf tissue.
3. Chop the tissue as fine as possible with a knife and chopping board.
4. Add the tissue to an ice-cold mortar containing 15 mL of grinding solution and grind the tissue to a paste.
5. Filter the ground up tissue solution through double-layered cheesecloth into a beaker, and squeeze the tissue pulp to recover all of the liquid suspension.
6. Transfer the green suspension to a cold 50-mL centrifuge tube and centrifuge for 1 minute at 200 xg at 4°C to pellet the unbroken cells and cell fragments.
7. Decant the supernatant into a clean centrifuge tube and centrifuge again for 7 minutes at 1000 xg to pellet the chloroplasts. Decant and discard the supernatant.
8. Resuspend the chloroplasts in 5 mL of cold suspension solution (or 0.35 M NaCl). Use a cold glass stirring rod to gently disrupt the packed pellet.
9. Enclose the tube in aluminum foil and place at 0°C to 4°C.
10. Using a hemacytometer, determine the number of chloroplasts per mL of suspension media.

Solutions

• Spinach leaves (fresh) suspension solution
• 0.33 M Sorbitol
• pH 7.6, adjust with NaOH
• 1 mM MgCl₂
• 50 mM HEPES
• 2 mM EDTA

Grinding Solution

• 0.33 M Sorbitol
• pH 6.5, adjust with HCl
• 4 mM MgCl₂
• 2 mM ascorbic acid (Vitamin C)
• 10 mM sodium pyrophosphate
• 0.35 M NaCl
• Bioreagents and Chemicals
• Sodium hydroxide
• Sodium pyrophosphate
• Sodium chloride
• Spinach leaves (fresh)
• Magnesium chloride
• Ascorbic acid
• Sorbitol
• HEPES
• Cheesecloth
• Hydrochloric acid
• EDTA

Protocol
The isolation procedure used in this protocol 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 for 5 minutes at 8000 xg to collect the chloroplasts. Remove the diluted suspension media and resuspend the chloroplasts in isotonic media (0.35 M NaCl or undiluted suspension buffer).