Wendel Lab
Department of Ecology, Evolution, and Organismal Biology
Iowa State University, Ames, IA 50011, USA
Materials and Reagents
1. Unless otherwise stated, all solutions were prepared in deionized water (18.2 M W conductivity).
2. Filter sterilization solutions using either 25 mm syringe filter (0.2 µm, nylon; Fisher Scientific, Houston, TX, USA; product number, 09-719C) or steritop (0.22 µm; Millipore Corporation, Billerica, Massachusetts, USA; product number, SCGPS02RE) depending on solution volume.
3. Mortar and pestle.
4. Extraction buffer: 100 mM Tris-HCl, pH 8.8, 10 mM (w/v) ethylenediamine tetra-acetic acid (EDTA), 900 mM (w/v) sucrose, 0.4% (v/v) 2-mercaptoethanol. Store at 4ºC. (Note: 2-mercaptoethanol should be added just before preparation of ‘phenol extraction buffer’).
5. Phenol buffered with Tris-HCl, pH 8.8 (Acros Organics, Morris Plain, NJ, USA; product number 327125000). Store at 4ºC. (Note: Prepared buffered phenol solution should be kept at -20ºC in 40 mL aliquots in 50 mL polypropylene tubes).
6. Phenol extraction buffer: Add equal volume of extraction buffer (plus phosphatase and protease inhibitors solution fromStep 4) and buffered phenol (fromStep 3) in a 15 mL Falcon tube and mix before use.
7. Ammonium acetate/methanol solution: 100 mM (w/v) ammonium acetate in 100% methanol. Store at 4ºC.
8. 80% (v/v) acetone in deionized water. Store at 4ºC.
9. 70% (v/v) ethanol in deionized water. Store at 4ºC.
10. Isoelectric focusing ( IEF) extraction solution: 8 M (w/v) urea, 2 M (w/v) thiourea, 4% (w/v) CHAPS, 2% (v/v) Triton X-100, and 50 (w/v) mM DTT. Store in single-use aliquots of 1 mL at -20ºC.
11. Balance and load shaking tray (Nutator; Becton Dickinson, Franklin Lakes, NJ, USA).
Methodology
1. Grind 250 mg of plant materials to a powder with liquid nitrogen in a mortar and pestle. (Note: Total protein extraction should be performed in the fume hood ).
2. Add 10 mL phenol extraction buffer and continue grinding for an additional 30 sec.
3. Transfer to a 15 mL Falcon tube and agitate on Nutator for 30 min at 4°C.
4. Centrifuge 30 min at 4000 rpm (4°C).
5. Transfer upper phase solution to a fresh 50 mL Falcon tube. (Note: Do not remove the white interface between the phenol and aqueous layer ).
6. Add 5 volumes of ammonium acetate/methanol solution (ice cold), vortex, and incubate at -20°C overnight to precipitate the phenol extracted proteins.
7. Centrifuge 30 min at 4000 rpm (4°C) to collect the precipitate.
8. Wash the pellet twice with ice-cold ammonium acetate/methanol solution, then twice with ice-cold 80% acetone solution, and fially with cold 70% ethanol solution. (Note: For the first resuspension, sonicate the sample. It is important to completely resuspend the pellet each time, by vortexing if necessary. Place the resuspended sample at -20°C for 20 min in between each wash).
9. Dry the final pellet (after removing the wash solution) 20 min at 37°C. (Note: Do not over-dry the protein pellet. A completely dry pellet is very difficult to resuspend in IEF extraction solution. Store at -80 ° C in acetone until further use ) .
10. Resuspend the final pellet in IEF extraction solution. (Note: Pipetting and/or vortexing at RT help in resuspending the pellet. If necessary incubate the sample for 30 to 60 min at RT with gentle agitation. Do not heat sample under any circumstances as this will lead to protein carbamylation ).
11. Centrifuge 15 min at 14000 rpm at RT.
12. Carefully transfer clear supernatant to a new 1.5 mL microfuge tube. Store in single-use aliquots at -80