GOLGI-VESICLE PREPARATION FROM PEA HYPOCOTYLS
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| PREPARE SOLUTIONS | |
| 1. 0.25 M Sucrose Solution: |
Mix 40 g of sucrose (0.25M), 50 mL of 1M KH2 PO4 , pH 6.65 (0.1M), 2.5 mL of 1M MgCl2 (5 mM), and dH2 O to 500 g. Filter through 0.45m. Add 1 m L/mL of 1M DTT (added right before use). Store at 4o C
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| 2. 0.5 M Sucrose Solution: |
Mix 80 g of sucrose (0.25M), 50 mL of 1M KH2 PO4 , pH 6.65 (0.1M), 2.5 mL of 1M MgCl2 (5 mM), and dH2 O to 500 g. Filter through 0.45m. Add 1 m L/mL of 1M DTT (added right before use). Store at 4o C
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| 3. 1.1 M Sucrose Solution: |
Mix 165 g of sucrose (0.25M), 50 mL of 1M KH2 PO4 , pH 6.65 (0.1M), 2.5 mL of 1M MgCl2 (5 mM), and dH2 O to 500 g. Filter through 0.45m. Add 1 m L/mL of 1M DTT (added right before use). Store at 4o C
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| 4. 1.3 M Sucrose Solution: |
Mix 190 g of sucrose (0.25M), 50 mL of 1M KH2 PO4 , pH 6.65 (0.1M), 2.5 mL of 1M MgCl2 (5 mM), and dH2 O to 500 g. Filter through 0.45m. Add 1 m L/mL of 1M DTT (added right before use). Store at 4o C
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| 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 dH2 O to 500 mL. Aliquot and store at -20o C
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| 6. 1M KH2 PO4 , pH 6.65 |
Mix 68 g of KH2 PO4 with 300 mL of dH2 O. pH to 6.65. Add dH2 O to 500 mL (Autoclave)
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| 7. 1M MgCl2 (500 mL): |
Mix 101.6 g of MgCl2 with dH2 O to 500 mL (Autoclave)
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| PROCEDURE | |
| NOTE: perform all steps at 4 o C | |
| 1. Grow pea seedlings (Pisum sativum var. Alaska) in moist vermiculite for ~7 days in the dark at 25o C | |
| 2. Cut ~1 cm segments from the elongating region of the hypocotyl (it should look like a hook) | |
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3. 40-70 g of tissue was homogenized by hand with razor blades in the presence of 1 volume of 0.5M Sucrose Solution |
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| 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 | |
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8. Centrifuge at 100,000 xg in a swinging bucket rotor for 100 minutes |
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| 9. Collect the 0.25M/1.1M interphase fraction, add 1 volume of dH2 O, 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 -70o C |
