Title: Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network Document date: 1991_12_2
ID: syyi2ysq_23
Snippet: A heat-stable protein fraction highly enriched in granins was prepared from [a 5 S]sulfate-labeled PC12 cells as previously described (Ross et al ., 1985) and dialyzed at 4°C against 4 mM MES, pH 7.2, containing 2 mM KCI . The heat-stable protein fraction was centrifuged for 1 h at 23,000 g to remove any granin aggregates . 50-pl aliquots of the supernatant were mixed with 250 Al of nonaggregative or aggregative milieu (final protein concentrati.....
Document: A heat-stable protein fraction highly enriched in granins was prepared from [a 5 S]sulfate-labeled PC12 cells as previously described (Ross et al ., 1985) and dialyzed at 4°C against 4 mM MES, pH 7.2, containing 2 mM KCI . The heat-stable protein fraction was centrifuged for 1 h at 23,000 g to remove any granin aggregates . 50-pl aliquots of the supernatant were mixed with 250 Al of nonaggregative or aggregative milieu (final protein concentration = 68 pg/ml) and incubated for 2 h at 0°C. Samples were centrifuged (23,000 g for 30 min) and pellets and supernatants were analyzed by SDS-PAGE followed by fluorography Carbonate TYeatment [IS] sulfate-labeled TON vesicles were prepared from control and xylosidetreated PC12 cells according to the standard procedure. Pelleted TGN vesicles derived from 1-2 ml of fractions 8-10 of the velocity gradient were resuspended in 1 ml of H2O. This and all subsequent manipulations were performed at 0-4°C. While stirring, 1 ml of 2x concentrated carbonate buffer (lx carbonate buffer consisted of: 0.1 M Na2CO3-NaHCO3, pH Chanat and Huttner Aggregation-mediating Sorting of Secretary Proteins 11A, 1.0 M KCI, 0.25 mg/ml saponin, 2 mM EDTA, 0.1 mM PMSF) was slowly added to the membrane suspension. After a 30-min incubation under stirring, membranes were centrifuged . This and all subsequent centrifugations were carried out at 130,000 g for 45 min. The supernatant was neutralized with 1 N HCl to pH 7.5 and subjected to acetone precipitation (70% (vol/vol) final concentration, 15 h at -20°C) before being analyzed by SDS-PAGE and fluorography Membranes were resuspended in carbonate buffer, stirred for 15 min, pelleted, and further washed for 15 min in 10 mM MES, pH 6.5, 2 mM EDTA . After centrifugation, the pellet was analyzed by SDS-PAGE followed by fluorography.
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