Plasma membrane see Protocols 425 and 426

As with ER and Golgi, the plasma membrane is often analysed along with other membrane compartments such as endosomes and the trans-Golgi network (TGN) during studies into membrane trafficking and cell signalling (see Chapter 5). Protocols 4.25 and 4.26 in this chapter, by contrast, are concerned primarily with the bulk (preparative) isolation of these membranes. There are two principal sources for bulk isolation of mammalian plasma membrane that have been used for studies of membrane composition, function and architecture: the human erythrocyte and rat liver. The human erythrocyte membrane in particular is easy to isolate by hypotonic lysis [55] and the lack of internal organelles means that density gradient fractionation is not required (Protocol 4.25). Bacterial limiting membranes also pose relatively few problems [56]. Structures such as desmosomes, tight junctions or an extensive cytoskeleton, effectively stabilize certain plasma membrane domains from polarized tissues against fragmentation during homog-enization. Thus, the contiguous membrane of rat liver or the brush border of intestinal

CHLOROPLASTS (SEE PROTOCOL 4.30)

epithelial cells may also be isolated relatively easily, in the form of sheets, which are rapidly sedimenting and easily separated from most other organelles and membrane vesicles. Protocol 4.26 describes the isolation of the contiguous membrane from rat liver [57].

Plasma membrane analysis using enzyme markers (Protocols 4.27-4.29)

Routine functional analysis of plasma membrane relies on enzyme assays, principally for Na+/K+-ATPase, 5'-nucleotidase, alkaline phosphatase or alkaline phosphodiesterase. There is considerable variation in levels of these enzymes in plasma membranes from different sources and in the case of cells from polarized tissues; they are often domain-specific. Thus, the Na+/K+-ATPase is readily measured in the human erythrocyte membrane, much less easily measured in the plasma membrane from cultured cells and considerably enriched in the basolateral domains (compared to apical domains) from polarized cells. Alkaline phosphatase, alkaline phosphodiesterase and 5'-nucleotidase are ubiquitous, highly enriched in apical domains and easier to measure than the Na+/K+-ATPase.

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