bis_104_pq_14_ans_spring_08

bis_104_pq_14_ans_spring_08 - b) Biochemical experiment:...

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BIS 104 PQ 14 spring 08 14. In an attempt to understand the underlying cellular mechanisms that cause symptoms of cystic fibrosis, investigators compared live cells taken from the bronchial epithelium of normal volunteers and from cystic fibrosis patients. First, they tested the hypothesis that cells from CF patients would lack the CFTR chloride channel protein in their plasma membranes. Describe two experimental approaches to test this hypothesis: one cytological procedure and one biochemical procedure. a) cytological experiment: Obtain a fluorescently labeled antibody specific for the external domain of the CFTR protein. Treat normal cells and cells from CF patients with this antibody. Wash away any unbound antibody and examine cells by fluor microscopy. Compare PM fluor between normal and CF cells
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Unformatted text preview: b) Biochemical experiment: Isolate plasma membrane fractions from both normal and CF cells. Run plasma membrane proteins out on an SDS-PAGE Transblot gel and probe blot with a labeled antibody specific for CFTR protein Compare lane with normal membrane proteins with lane with CF sample Lets suppose that the above hypothesis was proven to be correct. Describe what experiment you would do next to further our understanding of the role of CFTR protein in this disease. c) Experiment: Create a DNA construct containing sequence coding for normal CFTR protein linked to a reporter gene, such as GFP; link this to strong promoter. Transfect CF cells with this construct. Confirm expression of CFTR in PM of host cells and determine if this restores chloride efflux function. (gain of function)...
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bis_104_pq_14_ans_spring_08 - b) Biochemical experiment:...

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