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. 1997 Jan 27;136(2):287–297. doi: 10.1083/jcb.136.2.287

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Sucrose gradient fractionation of A-ALP/RS-ALP containing membranes. High speed pellet membrane fractions (100,000 g) were prepared from 100 OD units of SNY36-9A cells harboring either pSN55 (A-ALP) or pSN97 (RS-ALP). These membranes were fractionated by a 20–50% sucrose density step gradient. After centrifugation, 14 × 1–ml fractions were collected from the top of the gradient, and the proteins were precipitated by the addition of TCA. Equal percentages of each fraction were subjected to immunoblot analysis after SDS-PAGE to detect the following proteins: A-ALP/RS-ALP using an anti-ALP mAb (1D3-A10); Kex2p was used as a marker protein for TGN membranes and detected using affinity-purified polyclonal antibodies; anti-Vph1p mAb 10D7-A7-B2 was used to detect Vph1p as a marker protein for vacuolar membranes; and Pep12p, a marker protein for membranes of the prevacuolar compartment, was detected using affinity-purified polyclonal antibodies.