Protocol: Preparation of Plant Protein Extracts
- Cut plant tissue into pieces and grind in a mortar and pestle with liquid nitrogen to produce a fine powder. Keep the powder frozen.
- Just before use, prepare extraction buffer. Dissolve PMSF (phenylmethyl sulfonyl fluoride) in 100% ethanol to produce a 200 mM solution. Add PMSF, β-mercaptoethanol and one Complete Protease Inhibitor tablet (Roche) per 10 mL to a solution of 0.7 M sucrose, 0.5 M Tris, 30 mM HCL, 50 mM EDTA, 0.1 M KCl to produce the Hurkman extraction buffer (0.7 M sucrose, 0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCl , 2% (v/v) 2-mercaptoethanol and 2 mM PMSF with protease inhibitors).
- Place 10 mL Hurkman extraction buffer in a 50 mL Falcon tube. Add the desired amount of frozen plant tissue (~5g). Invert and vortex until powder appears to go into solution. Add 0.1g PVPP (polyvinylpyrrolidone) and vortex well.
- Incubate for 10 min at 4°C. Centrifuge at 3210 x g for 30 minutes at 4°C. Remove supernatant and transfer to a clean 50 mL Falcon tube.
- Add 10 mL of phenol saturated with 1 M Tris, pH 7.9. Vortex for 30 seconds. Let sit 30 minutes on ice, mixing tubes every 10 minutes.
- Separate the phases by centrifugation at 3210 x g, 30 minutes, 4°C. Carefully remove the top layer (phenol phase) and transfer to a clean 50 ml Falcon tube. Estimate volume of phenol phase.
- Re-extract with an equal volume of Hurkman extraction buffer. Transfer top layer to a clean Falcon tube.
- Precipitate proteins from the phenol phase by addition of 5 volumes of 0.1 M ammonium acetate in methanol. Vortex for 30 seconds and incubate at -20°C overnight.
- Wash the precipitate 1 time with 0.1 M ammonium acetate in methanol and 3 times with very cold acetone. Air dry the pellet.
- For LC/MS, dissolve the pellet in 25 mM ammonium bicarbonate. Centrifuge at 100,000 x g, 4°C for 1 hour. Carefully remove supernatant and store at 4°C. Perform protein assay.
- For 2-D gel analysis, solubilize the pellet in desired rehydration buffer by incubation for 1 hour at room temperature. Centrifuge at 100,000 x g, 18°C, for 1 hour. Remove supernatant and perform protein assay.
Grimplet J., et al. Proteomics (2009) 9, 2503-2528.