Protocols > BBY preparation

Protocol

1. Homogenise 100 g Spinach with 200 ml Prep. medium: (300 mM Sucrose 50 mM Sodium phosphate buffer pH 7.4, 5 mM MgCl₂). Add 0.1% BSA just before start to prepmedium. 3 x 5 seconds. Filter through 4x Cheesecloth.
2. Centrifuge 3000 rpm, 3 min. (SS-34) You will need a lot tubes at this stage. Try to be fast until this step, protease's are working on your proteins! Resuspend gentle with brush in Prep. medium: (300 mM Sucrose, 50 mM Sodium phosphate buffer pH 7.4, 5mM MgCl₂). Two pellets in one tube.
3. Centrifuge 3000 rpm for 10 minutes (SS-34).
4. Resuspend in 5 mM MgCl₂ and homogenise with glaspotter (pistil A). Fill tube (SS-34) half. Leave some minutes. Fill tubes with resuspending medium: (400 mM Sucrose, 50 mM MES pH 6.5, 15 mM NaCl).
5. Centrifuge 5000 rpm for 10 minutes.
6. Wash once by resuspending and homogenise in wash medium: (2 mM MES, 5 mM MgCl₂, 15 mM NaCl).
7. Centrifuge 5000 rpm for 10 minutes.
8. Resuspend in same buffer, to 100 ml. Measure the Chlorophyll concentration. Dilute sample to 2.67 mg Chl/ml with same buffer, check volume (for example 150 ml). Take out thylakoid sample for control. Leave on ice, dark and with slow steering for at least 30 minutes.
9. Add the 20% Triton X-100 solution, 1/3 of volume of thylakoids, slowly to the slow steering sample on ice. Start watch. (i.e. 150/3 = 50 ml).
10. After 20 minutes, add sample to centrifuge tube (SS-34), and after 25 minutes, make a slow speed centrifugation 10 000 rpm for 4 minutes.
11. Transfer supernatant to new tubes, and centrifuge at 4°C for 30 minutes at 19 500 rpm.
12. Discard the very green supernatant. Resuspend the PS II/BBY pellet in resuspention buffer: (400 mM Sucrose, 50 mM MES pH 6.5, 15 mM NaCl) or if higher yields are wonted in wash medium, homogenise with glashomogeniser. Centrifuge again for 30 minutes at 19 500 rpm.
13. Resuspend in resuspention buffer: (400 mM Sucrose, 50 mM MES pH 6.5, 15 mM NaCl), use spatula to get as high concentration as possible. Measure chlorophyll. Freeze in small aliquots at -80°C or N₂.
14. "God" BBY have Chl a/b of 1.8 (lower is better!). Yields: 1000 g Spinach --> 10% in mg Chl (100mg) of thylakoids. Keep cool during the prep, and avoid direct light.

Solutions

20% Triton X-100 medium, 200 g