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Protocols > Arabidopsis thaliana thylakoid extraction

Courtesy of Prof. Wolfgang Schröder, Umeå University.

All steps were done in a cold-room on ice, and in as dark conditions as possible. All the centrifugations were done in +4°C.
Put the centrifuges etc. in cooling before starting, and buffers the day before in cold-room.

Grind the leaves (≈20 g) in 170 ml of Grinding buffer. We use a normal kitchen blender (sharp blades!). Something like five 1.5 sec pulsations.
Filter through four layers of Miracloth/cheescloth in a 1L Erlenmeyer kept on ice.
Pour the filtrate in 30 mL centrifugation tubes.
Centrifug in JA 25.50, 1000 g x 3 min
Pour away the supernatant and resuspend the pellet gently (with a soft brush) with Wash buffer. Centrifug in JA 25.50, 1000 g x 3 min
Pour away the supernatant and resuspend the pellet to 20 ml with Shock buffer. Use glass homogenizer with pestle A, three times.
Centrifug in JA 25.50, 2000 g x 5 min
Pour away the supernatant and re-suspend the pellet in Stack buffer in as small volume of as possible (like 1.5ml). Homogenize properly, use glass homogenizer and pipet the thylakoids into aliquots.
Measure Chlorophyll
- Thylakoids normally Chl a/b of 3-3.5 (depending on growth light used

2 x 50 g = 100 g leaf ≈ 30 mg Chl Thylakoids
3 x 50 g = 150 g Arabidopsis leaf ≈ 60 mg Chl Thylakoids

Grinding buffer, 1 L

Just before use add 2 g of both BSA and sodium ascorbate in one liter of Grinding buffer.

Wash buffer, 500 ml

Shock buffer, 500 ml

20 mM Tricine-NaOH, pH 7.6		20 ml, 0.5 M stock
5 mM MgCl₂		2.5 ml, 1 M stock

Stack buffer, 250 ml