Glutamate Urea Mg2

Glutamate (+Mg2+)

Cu G

10 15 Fractions

5 10

Fractions

5 10

Fractions

5 10

Fractions

5 10

Fractions o a

Figure 2.1 Equilibrium density gradient analysis. Shown are the results of an equilibrium density gradient analysis carried out to determine the subcellular localization of Gaplp, the general amino acid permease, under different culture conditions (glutamate or glutamate transferred to urea). (Taken from Roberg et al., 1997.) The density gradient is 20%-60% sucrose with and without Mg2+. The upper row of panels shows, for each fraction, the sucrose concentration (open circles) and the relative levels of Gaplp-HA (hemagglutinin-tagged Gapl protein) as determined by Western analysis. The lower row of panels shows the relative levels of Pmalp (plasma membrane marker) and Sec├│lp (endoplasmic reticulum marker) as determined by Western analysis and the activity of GDPase (Golgi marker) as determined by enzymatic assay. The horizontal bars above the lower set of panels indicates the position in the gradient of each membrane type. Reproduced from The Journal of Cell Biology, 1997, by copyright permission of The Rockefeller University Press

tri O X

Vi subcellular component can be assayed (either biochemically or by Western analysis) to assess the purity of a sample. Examples of such marker enzymes can be found in Kreutzfeldt & Witt (1991), Griffin (1994), and are listed in Walker (1998).

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