|Contributor: || The Laboratory of William Heidcamp at Gustavus Adolphus University |
|This protocol describes a simple procedure for isolating chloroplasts from spinach leaves. |
| 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.
|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
| Spinach Leaves (Fresh) |