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Extra info for Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology
For a six-well tissue culture plate, label the cells with 2 ml of labeling solution (DMEM without methionine, containing 10% [v/v] dialyzed serum, and 400 &i of [35S]methionine) at 37” for 3 to 6 hr. 3. don a Centricon-10 (Amicon). 4. Mix the supernatant with 20 ~1 of beads coupled with anti-AP antibody for 30 min or a rotator at room temperature. 5. 1% (w/v) NP-40. Use ice-cold buffers and do this quickly. Spins are at 5000 rpm for 1 min in a microcentrifuge. 6. Add an equal volume of loading buffer and heat the sample for 2 min at 100”.
The determination of intracellular distribution in such cases usually relies on quantitating levels of the protein within subcellular fractions obtained by differential centrifugation of postnuclear supernatants. Using subcellular fractionation, we have been quantitating the steady state levels of chimeras comprising the transmembrane and cytoplasmic domains of proteins including P-selectin, sialyltransferase, tyrosinase, and the transferrin receptor (TmR), in which their lumenal domains have been METHODS IN ENZYMOLOGY, VOL.
Desnos, Y. Lichtenstein, V. -T. Horng, and R. B. Kelly, Methods Enzymol. 5'0(1998). 141 ANALYSIS OF TRAFFICKING WITH HRP CHIMERAS 51 10 15 Fraction number FIG. 2. Isolation of SLMVs from PC12 cells. PC12 cells, transiently expressing wild-type HRP-P-selectin, were homogenized in buffer A (see text) and PNS was centrifuged on the preformed S-25% glycerol gradients as described. The distribution of HRP activity (0) for wild-type HRP-P-selectin relative to that of the endogenous marker protein synaptophysin/ ~38 immunoreactivity is shown in (A) and (B).