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. 2007 Jul 6;6(9):1625–1634. doi: 10.1128/EC.00198-07

FIG. 1.

FIG. 1.

Characterization of microsomal membrane preparations. (A) Cell homogenates were prepared and subjected to differential centrifugation. Equal protein amounts (5 μg) of membrane pellets obtained at 3,000 × gav (P3), 9,000 × gav (P9), 20,000 × gav (P20), 40,000 × gav (P40), and 100,000 × gav (P100) were analyzed by SDS-PAGE and immunoblotting using antibodies against the marker proteins indicated. (B) The intactness of microsomal vesicles was assayed by protease protection. P100 membranes were incubated with trypsin (0.25 mg/ml) in the absence or presence of 0.5% Triton X-100 (TX-100). At the times indicated, protease digestion was terminated by addition of a trypsin inhibitor (1 mg/ml), and proteins were analyzed by SDS-PAGE and immunoblotting using antibodies against Wbp1p and Dpm1p. The integrity of microsomal membranes is demonstrated by the protection of Wbp1p from proteolysis in the absence of detergent.