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Isolation of Chloroplasts from Spinach Leaves
Contributor: The Laboratory of William Heidcamp at Gustavus Adolphus University
 
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
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
Spinach Leaves (Fresh)
Suspension Solution   0.33 M Sorbitol
pH 7.6 Adjust with NaOH
1 mM MgCl2
50 mM HEPES
2 mM EDTA
 
BioReagents and Chemicals
Spinach Leaves (Fresh)
Magnesium Chloride
Ascorbic Acid
Sorbitol
HEPES
Cheesecloth
Hydrochloric Acid
EDTA
Sodium Hydroxide
Sodium Pyrophosphate
Sodium Chloride
 
Protocol Hints