PROTEIN PREPARATION FROM PLANT TISSUES |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. Cold-H buffer (500 mL): | Mix 25 mL of 1M Hepes-KOH pH 7 (50 mM), 44.8 g of sucrose (0.25 M), 1.0 mL of 0.5M EDTA (1 mM), and add dH2O to 500 mL. Store at 4oC. NOTE: some enzymes require cations for activity. If activity is desired for such enzymes, exclude EDTA or use another kind of chelating agent (for a different cation, such as EGTA). |
| 2. Cold-S buffer (50 mL): | Mix
2.5 mL of 1M Tris, pH 7.5 (50mM), 4.48 g of sucrose (0.25M), 100 mL of 0.5M EDTA (1mM), and add dH2O to 50 mL |
| 3. 1M HEPES-KOH, pH 7 (1 L): | Add 238.3 g of HEPES, pH to 7 with KOH, and add dH2O to 1 L |
| 4. Resuspension buffer (100 mL): | Mix 5 mL of1M Hepes-KOH, pH 7 (50 mM), 10.3 g of sucrose (0.3M), MgOAc2 to 5mM, KOAc to 0.1 M, and add dH2O to 100 mL, Store at 4oC |
| 5. Cold-T buffer (500 mL): | Mix 25 mL of Tris-HCl pH 7.5 (50mM), 44.8 g of sucrose (0.25 M), 1.0 mL of 0.5M EDTA (1 mM), and add dH2O to 500 mL, Store at 4oC |
| 6. Detergent extraction buffer (100 mL): | Mix 5 mL of Hepes-KOH, pH 7 (50 mM), 5 mL of 20% Triton X100 (1%), 1.0 g of sodium-lauryl sarcosine (1%), add dH2O to 100 mL, 10 mL of 0.1 M DTT (added fresh) per 1 mL buffer, Store at 4oC |
| PROCEDURE | |
| NOTE: the tissue source and protein of interest are very important considerations before starting an extraction. These conditions have been used because they have given good results with the proteins I have worked on. These may NOT be appropiate for other applications | |
| NOTE: If plants were grown in liquid media: Grasp the plants by the leaves with forceps into a large glass petri dish full of dH2O. Swirl plants to aid in separating the roots from the leaves. Cut roots away from leaves with scissors or just by pulling | |
| NOTE: If plants were grown in agar: "peel" off plants from agar carfully using forceps to limit the amount of breaking of the roots | |
| Work must be performed on ICE at all times | |
| 1. Grind fresh tissue (up to 1 g) to a fine powder using a mortar and pestle under liquid nitrogen | |
NOTE: using liquid nitrogen tends to result in the loss of some tissue. If tissue amounts are very small, use extra caution (let the liquid nitrogen evaporate before grinding) |
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| OPTION: use a cordless mixer (Kontes, Cat#749540-0000) with a disposable mixer to grind the tissue in an eppendorf tube in the presence of liquid nitrogen. Small amounts of tissue can be prepared this way with little or no loss of tissue. Scale down volumes accordingly | |
| BEST: use a glass dounce homogenizer with an abrasive surface (clearance between pestle and tube ~0.15mm) | |
| NOTE: Arabidopsis stems require a lot more grinding than any other tissue, so if possible grind them in a mortar instead | |
| 2. Add 1 mL of Cold (H, T or S) buffer per 1g of tissue*, then 10 mL of 0.1 mM PMSF. Mix with pestle and pour into 15 mL centrifuge tube | |
*NOTE: every tissue is different. This protocol works for soft tissues like leaves and flowers. Other tissues like roots and stems require more buffer |
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| BEST: in addition to the PMSF, other protease inhibitors should be added. A good cocktail of protease inhibitors is Complete (from Roche). This cocktail comes as a tablet that can be dissolved in 10 mL of buffer and provides good inhibition against 5 of the main proteases (at pH 7.8) | |
| OPTIONAL: for denaturing conditions, 1 mL of 1M DTT may be added at this point | |
NOTE: if many samples need to be analyzed or there are many samples, tubes may rocked at 4oC for up to 1 h (depending on the protein in question, this may be too much) |
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| 3. Centrifuge 1,000g for 5 mins at 4oC | |
| 4. Collect supernatant (S1) with a pipette and transfer to a new tube (store at -20oC) | |
| 5. Place S1 in a 1 mL clear Beckman thick ultracentrifuge tube. Centrifuge at 45 krpms (150,000 g) for 2 hrs (4oC) | |
| 6. Collect supernatant (S150) and save, rinse the pellet (P150) twice with 5 mL Cold buffer | |
7. Resuspend the pellet (P150) by pipetting with 1 mL of Resuspension buffer OR Detergent extraction buffer OR the pellet is extracted on ice with 1 mL of 0.1 M Na2CO3, pH 11.5 for 30 mins. After centrifugation (12,000 g for 10 min), the pellet is resuspended in 0.5 mL 10 mM Hepes-KOH, pH 7 |
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| NOTE: the Na2CO3-washed membrane pellet can not be solubilized | |
NOTE: resuspending the P150 takes time. If all fail try incubating for 5 min at 37oC |
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