BIS 104 - SS II 2011 Midterm 1 (edwards)

BIS 104 - SS II 2011 Midterm 1 (edwards) - EXAM PAGE 2 1a N...

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EXAM PAGE 2 1a. N = 3 bands: from top of gel in descending order 210kD; 95kD; 25kD CF = 3 bands: from top of gel in descending order 190K; 95kD; 25kD 1b. N = same as above CF = 3 bands: from top of gel in descending order 190kD; 95kD; 25kD 190kD band has 10-fold more intense CB staining than 210kD in N 1c. N (ion wash): 210kD; 95kD; 25kD band absent N (trypsin): 210kD absent; 95kD; 25kD absent; new band at ~20kD is cytoplasmic domain of CFTR (200 amino acids) CF (ion wash) 190kD; 95kD; 25kD absent CF (trypsin) same as above
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PAGE 3 NAME ________________________ 1d. Describe how you would use an immunochemical procedure to further test your hypothesis that the CFTR protein is on the surface of normal cells, but not on the surface of mutant CF cells. (10 pts) EXPERIMENT (list steps in chronological order; a control is not required) a. Obtain pure live populations of both WT and mutant cells. b. Expose cells to a fluorescently labeled antibody that is specific for binding to the extra-cellular domain of CFTR. c. After sufficient time for binding, wash away any unbound antibody. d. Examine both cell populations by fluorescence microscopy and compare the PM fluorescent intensity. RESULTS: If hypothesis is correct: PM of WT will show relative bright immunofluorescence. PM of mutant cells will show little or no fluorescence. 1e. Assuming that CFTR is the only integral membrane protein in these cells, what additional information about the CFTR protein in normal cells could you obtain by making freeze-fracture replicas of the cells and examining these by scanning electron microscopy (SEM)? This technique would: - indicate which membrane leaflet (inner vs outer) has the stronger association with the protein. - indicate the relative abundance of the CFTR protein in the PM (e.g., many protein copies vs few copies) - indicate the pattern of distribution of the protein (e.g., diffuse vs clustered)
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PAGE 4 NAME _____________________________ 2. You hypothesize that mutant S752P cells contain a low endogenous level of cytoplasmic VEGFA protein, but they fail to initiate new synthesis of VEGFA when stimulated by vitronectin.
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BIS 104 - SS II 2011 Midterm 1 (edwards) - EXAM PAGE 2 1a N...

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