Plant cell protein extraction for gel-free protein expression study
1. Grind plant in liquid nitrogen to a fine powder
2. Add 1.5 ml methanol to the pellet, briefly vortex and place at -20 ºC for 10 min
3. Centrifuge the suspension at 16,000 x g for 5 min at 4 ºC
4. Remove the supernatant with a pipette
5. Add 1.5 ml methanol to the pellet, briefly vortex and place at -20 ºC for 10 min
6. Centrifuge the suspension at 16,000 x g for 5 min at 4 ºC
7. Remove the supernatant with a pipette
8. Add 1.5 ml acetone to the pellet, briefly vortex and place at -20 ºC for 10 min
9. Centrifuge the suspension at 16,000 x g for 5 min at 4 ºC
10. Remove the supernatant with a pipette
11. Dry the pellet: air dry 5 -10 min or SpeedVac 30 -60 seconds
12. Weight the dried sample to determine the plant tissue mass
13. Add 0.1% RapiGest (Waters, Catalog # 186001861) (4 µl per mg) to the dried sample. Incubate 15 min at 22 ºC (Temperature must be between 15 – 30 ºC). (If you have Bead-beater, you can use Bead-Beater in this step).
14. Centrifuge the suspension at 16,000 x g for 30 min at room temperature
15. Collect and save the supernatant (total protein sample)
16. Total protein test
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