Protocols > Lumen extraction from Arabidopsis thaliana

Protocol

1. Harvest leaf in 20 g batches the evening before the extraction. Put in plastic bags, remove air and seal bags. Keep bags in cold room overnight. Best total amount to use is 100-200 g of leaf.
2. Homogenize (170 ml Extraction buffer to 20 g leaf) 1 x 5 seconds.
3. Filter through four layers of cheesecloth/nylon mesh (pore size 20-22 µm).
4. Centrifuge at 1000 g for 2 min.
5. Suspend pellet gently (use a brush) in Wash buffer, and pool 2 tubes in one.
6. Centrifuge at 1000 g for 2 min.
7. Resuspend ”intact” chloroplasts in a small of volume resuspension media, and pool all tubes in one. Measure the chlorophyll concentration. Dilute sample with Pyrophosphate buffer to a final concentration of 0.2 mg chlorophyll/ml. Homogenize in a 40 ml-glass homogenizer pistil A. This chlorophyll concentration should remain constant during all subsequent thylakoid washes.
8. Centrifuge at 7500 g for 5 minutes.
9. Wash twice with 10 mM sodium Pyrophosphate buffer (pH 7.8), and centrifuge at 7500 g for 5 min.
10. Wash twice with Tricin buffer (2 mM tricin, pH 7.8, 300 mM sucrose), and centrifuge at 7500 g for 5 min.
11. Wash twice with Yeda press buffer and centrifuge at 7500 g for 5 minutes.
12. Pool all tubes into one, resuspend in as little volume of Yeda press buffer as possible. Measure the chlorophyll concentration and then adjust it to 3-4 mg chlorophyll/ml.
13. Yeda press once with 100 bars of pressure.
14. Ultracentrifuge at 200 000 g for 2 x 1 hour.

Solutions

Pyrophosphate wash buffer, 250 ml
(shock medium to break chloroplasts)

Yeda press buffer, 250 ml