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 4C 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 4C (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

Determination of Chlorophyll Content in Chloroplasts Contributor: The Laboratory of William Heidcamp at Gustavus Adolphus University

Overview This protocol describes how to determine the chlorophyll content of chloroplasts using a spectrophotometric assay. Procedure 1. Isolate Chloroplasts from spinach leaves (See protocol for preparation

of chloroplasts from spinach leaves). 2. Pipet 1 ml of a chloroplast suspension into a 15 ml centrifuge tube and add 8 ml of Acetone. Mix thoroughly. 3. Add 1 ml of ddH2O, mix again and centrifuge at 1,000 X g for 3 to 5 minutes. 4. Measure the absorbance of the sample from Step 3 on a spectrophotometer at 652 nm (See Hint #1). 5. Calculate the amount of total chlorophyll in 1 ml of the Chloroplast suspension. The amount of chlorophyll is given by the equation: mg chlorophyll per ml = Absorbance / 34.5

BioReagents and Chemicals Acetone