Protocols

protein extraction for isoelectric focusing Phenol extraction followed by methanolic ammonium acetate precipitation

Posted on Thursday, December 04, 2003

Description
Phenol extraction followed by methanolic ammonium acetate precipitation – an effective protocol for sample preparation from protein-poor, recalcitrant tissues such as plants

Procedure
1. Grind 1 g of fresh tissue to a powder with liquid nitrogen in a mortar and pestle.
2. Add 2.5 mL of Tris pH8.8 buffered phenol and 2.5 mL of extraction media (0.1 M Tris-HCl pH 8.8, 10 mM EDTA, 0.4% 2-mercaptoethanol, 0.9 M sucrose) and continue grinding for an additional 30 sec in a fume hood. Alternatively, transfer to a 15 or 50 mL Falcon tube and homogenize in polytron homogenizer for one minute.
3. Transfer to Falcon tube and agitate for 30 min at 4 C.
4. Centrifuge 10 min at 5000 g, 4 C.
5. Remove phenol phase (should be top phase) and back-extract aqueous phase with 2.5 mL + 2.5 mL of extraction media and phenol by vortexing. Centrifuge and combine with first extraction.
6. Precipitate phenol extracted proteins by adding 5 volumes of 0.1 M ammonium acetate in 100% methanol (stored at –20 C) to phenol phase.
7. Vortex and incubate at –20 C for at least 1 h or overnight. Collect the precipitate by centrifugation (20 min, 20,000 g, 4 C).
8. Wash the pellet 2X with 0.1 M ammonium acetate in methanol, 2X with ice-cold 80% acetone and finally 1X with cold 70% ethanol . Completely resuspend the pellet each time with vortexing and if necessary, sonication (this usually takes longer the first time). Place the resuspended sample at –20 C for at least 15 min between each wash. You can store the last suspended pellet in 80% acetone at –20 C until ready for IEF, or dry the last pellet under nitrogen (or at 37 C for 15 min) and store at -20 C.
9. Resuspend final pellet in 0.5-1 mL of IEF extraction solution (8 M urea, 2 M thiourea, 2% CHAPS, 2% Triton X-100, 50 mM DTT, 0.5% pH 3-10 ampholytes) by pipetting and vortexing at 25-30 C. Incubate sample for 1 h at room temperature with agitation. Do not heat sample under any circumstances as this will lead to carbamylation of proteins.
10. If protein quantitation is necessary, precipitate protein sample with TCA or acetone prior to performing Bradford or Lowry assay as detergents and reducing agents interfere with these assays.


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Submitted by: clamhankar