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        Yeast Protein Isolation "Yaffe-Schatz"酵母总蛋白提取

        互联网

        1946

        Adapted from Yaffe,M.P.and Schatz,G.,PNAS,1984,81,4819-4823.

        1.Grow 25mls yeast cells to 5x10E6.

        2.Sequentially spin down cells in a 15 ml polypro tube.

        3.Wash cells with 1ml ice cold ddH2O,transferrring to an eppy tube.

        4.Recon.cells in 1 ml ice cold ddH2O containing 100μg/ml PMSF.

        5.Add 150μl ice cold 2N NaOH 8% 2-ME (for 1 ml 400μl 5N NaOH 600μl ddH2O 80μl 2-ME).Mix by inverting tube several times.Inc.on ice for 10 min.

        6.Add 150μl ice cold 50% TCA,mix by inverting tube several times.Inc.on ice for 10 min.

        7.Spin 2 min.in microfuge.Wash pellet with 1ml.ice cold acetone,spin 2 min.and aspirate off the supernatant.Dry pellet.

        8.Resuspend pellet in 100μl sample buffer: 500μl 3X Sample buffer pH6.8 500μl ddH2O 12.5μl 2-ME 25μl 1M TRIS base 100μg PMSF dab of bromphenol blue.You may have to work at this,I've heard a wire homogenizer does the trick.

        9.Heat 95℃ x 2-5 min.Try loading 10μl on the gel,may need more.

        Note: Make PMSF at 10mg/ml in isopropanol and store at -20℃.

        •3X Sample Buffer:

        •0.2 M Tris pH6.8

        •6%SDS

        •30% Glycerol

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