Figure 5

Optimization of PEG/Dextran concentrations to be used for two phase affinity partitioning. A. Immunoblot analysis of partitioning of Nervana and BiP into PEG (P) and Dextran (D) at different concentrations of PEG/Dextran (from 5.4% PEG/Dextran to 6.9% PEG/Dextran). At concentrations above 6.0-6.3% Nervana partitions into the dextran phase preferentially while at 6.0% and below Nervana is found in both phases. BiP behaves identically. The significant drop in microsome partitioning into dextran seen between 5.7% and 6.3% is seen in all preparations but can be variable in extent, as illustrated in B. B. The same experiment performed for a range of percentages from 5.7-6.0% PEG/Dextran reveals a fairly abrupt transition between 5.8% and 5.9%. While a majority of both markers is seen in the PEG fraction at 5.7% and in the Dextran phase at ≥ 6.3%, the precise fractionation behaviour in the transition zone is somewhat variable. The Nrv and BiP blots shown in this panel are from different preparations to illustrate this phenomenon (see text for further discussion).