Isolation of Chloroplasts from Spinach Leaves

Overview

This protocol describes a simple procedure for isolating chloroplasts from spinach leaves.   Procedure

1. Prepare an ice bath and pre-cool 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 de-veined 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 X g 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 1,000 X g 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 to 4°C (see Hint #1).

10. Using a hemacytometer, determine the number of chloroplasts per ml of suspension media.

11. To determine the chlorophyll content see Protocol on Determination of Chlorophyll Content in Chloroplasts.

  Solutions

Suspension Solution		0.33 M Sorbitol pH 7.6 Adjust with NaOH 1 mM MgCl2 50 mM HEPES 2 mM EDTA
Grinding Solution		pH 6.5 Adjust with HCl 0.33 M Sorbitol 4 mM MgCl2 2 mM Ascorbic Acid (Vitamin C) 10 mM Sodium Pyrophosphate
0.35 M NaCl
Spinach Leaves (Fresh)

  BioReagents and Chemicals Cheesecloth
EDTA
Hydrochloric Acid
Sodium Hydroxide
Sodium Pyrophosphate
Sodium Chloride
Spinach Leaves (Fresh)
Magnesium Chloride
Ascorbic Acid
Sorbitol
HEPES 

  Protocol Hints

1. 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 8,000 X g to collect the chloroplasts. Remove the diluted suspension media and resuspend the chloroplasts in isotonic media (0.35 M NaCl or undiluted Suspension Buffer).