Golgi-Vesicles preparation from pea etiolated hypocotyls

GOLGI-VESICLE PREPARATION FROM PEA HYPOCOTYLS
	Last Update: December 2006
	Based on Munoz P, Norambuena L, Orellana A (1996) Evidence for a UDP-glucose transporter in Golgi apparatus-derived vesicles from pea and its possible role in polysaccharide biosynthesis. Plant Physiol 112: 1585-1594

PREPARE SOLUTIONS
1. 0.25 M Sucrose Solution:	Mix 40 g of sucrose (0.25M), 50 mL of 1M KH₂PO₄, pH 6.65 (0.1M), 2.5 mL of 1M MgCl₂ (5 mM), and dH₂O to 500 g. Filter through 0.45m. Add 1mL/mL of 1M DTT (added right before use). Store at 4^oC
2. 0.5 M Sucrose Solution:	Mix 80 g of sucrose (0.25M), 50 mL of 1M KH₂PO₄, pH 6.65 (0.1M), 2.5 mL of 1M MgCl₂ (5 mM), and dH₂O to 500 g. Filter through 0.45m. Add 1mL/mL of 1M DTT (added right before use). Store at 4^oC
3. 1.1 M Sucrose Solution:	Mix 165 g of sucrose (0.25M), 50 mL of 1M KH₂PO₄, pH 6.65 (0.1M), 2.5 mL of 1M MgCl₂ (5 mM), and dH₂O to 500 g. Filter through 0.45m. Add 1mL/mL of 1M DTT (added right before use). Store at 4^oC
4. 1.3 M Sucrose Solution:	Mix 190 g of sucrose (0.25M), 50 mL of 1M KH₂PO₄, pH 6.65 (0.1M), 2.5 mL of 1M MgCl₂ (5 mM), and dH₂O to 500 g. Filter through 0.45m. Add 1mL/mL of 1M DTT (added right before use). Store at 4^oC
5. 2X STM buffer (500 mL):	Mix 80 g of sucrose (0.5M), 1 mL of 1M MgCl₂ (2mM), 10 mL of 1M Tris-HCl, pH 7.5 (20mM), and dH₂O to 500 mL. Aliquot and store at -20^oC
6. 1M KH₂PO₄, pH 6.65	Mix 68 g of KH₂PO₄ with 300 mL of dH₂O. pH to 6.65. Add dH₂O to 500 mL (Autoclave)
7. 1M MgCl₂ (500 mL):	Mix 101.6 g of MgCl₂ with dH₂O to 500 mL (Autoclave)

PROCEDURE
NOTE: perform all steps at 4 ^oC
1. Grow pea seedlings (Pisum sativum var. Alaska) in moist vermiculite for ~7 days in the dark at 25^oC
2. Cut ~1 cm segments from the elongating region of the hypocotyl (it should look like a hook)
3. 40-70 g of tissue was homogenized by hand with razor blades in the presence of 1 volume of 0.5M Sucrose Solution
4. Once tissue is finely chopped, homogenize for 3 minutes in a mortar
5. Filter homogenate through Miracloth and centrifuge for 5 mins at 1,000 xg
6. Load supernatant on top of an 8 mL 1.3M Sucrose Solution cushion and centrifuge at 100,000g in a swinging bucket rotor for 90 minutes
7. Remove the upper phase without disturbing the interphase and then add 15 mL of 1.1M Sucrose Solution, followed by 5 mL of 0.25M Sucrose Solution
8. Centrifuge at 100,000 xg in a swinging bucket rotor for 100 minutes
9. Collect the 0.25M/1.1M interphase fraction, add 1 volume of dH₂O, mix slightly, and centrifuge at 100,000 xg in a fixed rotor for 50 minutes
10. Discard supernatant. Resuspend pellet using 2 mL of 1X STM buffer and a dounce homogenizer
11. Aliquot Golgi vesicles into 110 mL fractions and store at -70^oC