Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis

Abstract

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a geater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when mem (8,20). SDS givesgood solubilization ofmembrane proteins (1), but because of its anionic nature, proteins solubilized in SDS cannot be applied directly to isoelectric focusing gels. In this paper, we examine four methods of membrane solubilization, two of which include SDS, for preparation of samples for isoelectric focusing. Solubilization ofa plasma membrane-enriched fraction from barley roots was used to assess the electrophoretic separations ofthese protein preparations on silver stained 2D gels.MATERIALS AND METHODS Plant Material. Seeds of barley (Hordeum vulgare L. cv California Mariout 72) were sown on moist cheesecloth above aerated Johnson's medium (10) and grown at 22°C in the dark for 7 d.Membrane Preparation. Microsomal suspensions were obtained from barley roots as previously described and the plasma membrane-enriched fraction was isolated based on the distribution of microsomal membranes on linear sucrose gradients (9). Microsomal suspensions were applied to discontinuous sucrose gradients consisting of 10 ml of 22% (w/w), 13 ml of 30% (w/ w), and 13 ml of 40% (w/w) sucrose in 1 mM DTT and 1 mM EDTA adjusted to pH 7.2 with Tris. The gradients were centrifuged for 2 h at 80,000g and the plasma membrane-enriched fraction was collected with a syringe by puncturing the centrifuge tube at the 30/40% sucrose interface. The fraction was washed with 150 mM KCI and pelleted as previously described (9).Solubilization of Membrane Proteins. The centrifuge tubes containing membrane pellets were inverted on ice for 10 min and excess supernatant removed before addition ofsolubilization buffer. Four methods were used to solubilize the plasma membrane-enriched fraction; all solutions containing ampholytes had 1.6% pH range 5 to 7 and 0.4% pH range 3.5 to 10.

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