- Prepare an ice bath and precool all glassware to be used (including a
mortar and pestle).
- Select several fresh spinach leaves. Remove the large veins by tearing them
loose from the leaves. Weigh out 4 g of deveined leaf tissue.
- Chop the tissue as fine as possible with a knife and chopping board.
- Add the tissue to an ice-cold mortar containing 15 mL of grinding solution
and grind the tissue to a paste.
- Filter the ground up tissue solution through double-layered cheesecloth
into a beaker, and squeeze the tissue pulp to recover all of the liquid
- 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
- 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
- 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.
- Enclose the tube in aluminum foil and place at 0°C to 4°C.
- Using a hemacytometer, determine the number of chloroplasts per mL of
- Spinach leaves (fresh) suspension solution
- 0.33 M Sorbitol
- pH 7.6, adjust with NaOH
- 1 mM MgCl2
- 50 mM HEPES
- 2 mM EDTA
- 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
- Hydrochloric acid
The isolation procedure used in this protocol leaves the chloroplast
membrane intact. If you wish to study the enzymes for
the chloroplasts and rupture the outer membranes. To rupture the outer
membranes, resuspend the chloroplasts in diluted suspension solution
Immediately centrifuge the chloroplast suspension for 5 minutes at 8000
the chloroplasts. Remove the diluted suspension media and resuspend
the chloroplasts in isotonic media (0.35 M NaCl or undiluted suspension buffer).