35S-METHIONINE LABELING OF PROTOPLASTS |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. BSA coated plates: | coat plates with GB + 2% BSA, shaking for at least 1 hr |
| 2. GB: | Mix 3.2 g of Gamborgs B5 + minimal organics, add glucose to 0.44 M , 1 mg of 2,4 D, and Mes to 3 mM. pH to 5.7 (Autoclave) |
| 3. Lysis buffer: | Prepare 50 mM HEPES, pH 7.5, 10 mM KOAc, 1 mM EDTA, and 0.4 M sucrose (Autoclave) |
| PROCEDURE | |
| 1. Protoplast cells as described in Protoplast preparation (from plant tissue) | |
| 2. Pellet protoplasts and resuspend in GB at ~1x106 protoplasts/mL | |
| Note: from Arabidopsis suspension cells, 1 mL of PCV (pack cell volume) is roughly 4-6x106 cells | |
3. Aliquot 1 mL of protoplasts into BSA-coated wells (at least 6 wells per experiment) |
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| 4. Add 10 mL of 35S Met/Cys (~100 mCi) and shake in the dark ON at ~50 rpms | |
| 5. Collect cells in an eppendorf, spin for ~15 seconds and discard supernatant | |
| 6. Resuspend pellets in 4 mL of lysis buffer. Transfer to 15 mL tube. Lyze cells by pipeting up and down ~>8 times through a >25 G needle attached to a 3 mL syringe | |
| 7. Aliquot lysate into eppendorf tubes and spin for ~30 secs. Transfer cleared lysate to a new tube. Save pellet | |