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Publisher Summary This chapter discusses the transport of protein between endoplasmic reticulum and golgi compartments in semiintact cells. Preparation of cells that actively transport VSV G protein are most thoroughly characterized using CHO cells and CHO cytosol. However, there is considerable variability in transport using different cell lines or conditions. Where indicated in the procedures, measurement of transport using different marker proteins, or the use of different cell lines or cytosol preparations, may need to be optimized to obtain maximal efficiencies of transport. Extensive fragmentation and lysis of the ER (particularly in the case of strongly adherent cells) generally result in reduced transport. In particular, perforation conditions can lead to the release of soluble marker proteins from the ER during preparation of semiintact cells. In contrast, poor perforation leads to a transport that is efficient, but cytosol independent. In the latter case, cytosol dependence can be enhanced by gently homogenizing the semiintact cells with a loose-fitting pestle in a glass Dounce (10-20 strokes) prior to washing. |
