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BIS104 Edwards F06 MT1 - Page 4

BIS104 Edwards F06 MT1 - Page 4 - 4 Probe the blots with an...

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d. Describe how you could use an immunocytochemical procedure to further test your hypothesis that CFTR protein is on the surface of normal cells but not on the surface of the mutant CF cells. Experiment: 1. obtain pure populations of both cell types. 2. Using direct or indirect immunofluorescent technique, treat each cell type with a fluorescently labeled antibody directed against the CFTR protein. 3. Wash away any unbound antibodies. 4. Examine stained cells by fluorescence microscopy and compare the intensity of fluorescent emission in the normal cells vs the mutant cells. Results: Normal cells will show an intense positive membrane staining (fluorescence). CF cells will be negative . An alternative approach that is not as direct, but still valid: EXPERIMENT: 1. Obtain pure populations of both cell types. 2. Isolate and purify the plasma membranes from both cell types. 3. Run the PM proteins on an SDS-PAGE system and then transblot.
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Unformatted text preview: 4. Probe the blots with an enzyme-linked antibody that is specific for the CTFR protein; develop the blot and compare staining pattern of PM proteins from normal vs CF cells. RESULTS: Plasma membrane extract from normal cells will give a single positive band corresponding to a mol wt of ~ 210kd. There will be no band in the transblot from CF cells. e. What additional information about the CFTR protein in normal cells could you obtain by making f reeze-fracture replicas of the cells and examining these with electron microscopy? This procedure would reveal which side of the plasma membrane (inner vs outer leaflet) the CFTR protein was embedded in (bumps vs dimples). It would also allow one to see whether the protein was arranged in clusters, or distributed diffusely around the cell, or if it was localized to one region of the cell. Demo...
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