7/30/2019 Isolation of Chloroplasts From Spinach Leaves

1/3

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 fromspinach 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 tearingthem 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 andcell fragments.

7. Decant the supernatant into a clean centrifuge tube and centrifuge againfor 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 (or0.35 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

7/30/2019 Isolation of Chloroplasts From Spinach Leaves

2/3

of Chlorophyll Content in Chloroplasts.

Solutions

Grinding Solution 0.33 M SorbitolpH 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 HEPES2 mM EDTA

BioReagents and Chemicals

Spinach Leaves (Fresh)

Magnesium Chloride

Ascorbic Acid

SorbitolHEPES

Cheesecloth

Hydrochloric AcidEDTA

Sodium Hydroxide

Sodium Pyrophosphate

Sodium Chloride

Determination of Chlorophyll Content in Chloroplasts

Contributor: The Laboratory of William Heidcamp at GustavusAdolphus University

Overview

This protocol describes how to determine the chlorophyll content ofchloroplasts using a spectrophotometric assay.

Procedure

1. Isolate Chloroplasts from spinach leaves (See protocol for preparation

7/30/2019 Isolation of Chloroplasts From Spinach Leaves

3/3

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 5minutes.

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