skip to main content
×

Inclement Weather

All classes meeting today, Jan. 23, at 10 a.m. or later will convene at their regularly scheduled times. Check back for important, ongoing University updates or changes to campus operations.
Media Advisories Statements

Protocol: Preparation of Plant Protein Extracts

  1. Cut plant tissue into pieces and grind in a mortar and pestle with liquid nitrogen to produce a fine powder. Keep the powder frozen.
  2. 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).
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. Re-extract with an equal volume of Hurkman extraction buffer. Transfer top layer to a clean Falcon tube.
  8. 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.
  9. Wash the precipitate 1 time with 0.1 M ammonium acetate in methanol and 3 times with very cold acetone. Air dry the pellet.
  10. 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.
  11. 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.

Take the next step...