Protocols

protein extraction from plant tissues for isoelectric focusing: SDS extraction followed by acetone precipitation

Posted on Thursday, December 04, 2003

Description
SDS extraction followed by acetone precipitation – simple extraction protocol that does not require phenol. Recommended start protocol for whole tissue extractions.

Procedure
1. Grind 1 g of fresh tissue to a powder with liquid nitrogen in a mortar and pestle.
2. Add 5 mL of extraction media (0.175 M Tris-HCl, pH 8.8, 5% SDS, 15% glycerol, 0.3 M DTT) directly to mortar and continue grinding for an additional 30 sec.
3. Filter homogenate through two layers of miracloth into a 50 mL Falcon tube at room temperature.
4. Immediately add 4 volumes of ice cold 100% acetone to filtered homogenate, mix by vortexing and place at -20 C for at least one hour to precipitate proteins.
5. Centrifuge at 5000 g for 15 min to collect precipitated protein, decant supernatant.
6. Gently blot residual acetone from container with Kimwipe and then wash pellet in 15-20 mL of cold 80% acetone. Be sure to thoroughly break-up pellet by pipetting, vortexing or sonication.
7. Repeat steps 5 and 6.
8. Collect final protein precipitate by centrifugation at 5000 g for 15 min and dry pellet by inverting on Kimwipe for 15 min at 37 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. Centrifuge for 10 min at 12000 g and use supernatant to rehydrate IPG strips.
11. 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