Frye_and_Edidin_highlights_ans

Frye_and_Edidin_highlights_ans - Frye and Edidin highlights...

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Frye and Edidin highlights Describe the general outline of the experimental strategy used to demonstrate the fluid membrane. - Take two cells from different organisms (mouse and human) and fuse together with fusogen to make Heterokaryon – mark with antibody markers with florescent tags. Check for flourecent tags then if the two colors mix you get a mosaic meaning its fluid use of two different cell lines mouse cIID and Human Va-2 doesn’t die after 50 generations method to fuse the cells - what does sendai virus do? be able to picture how this works it fuses the cells time course of the experiment (fusion, incubation and then antibody staining) - Mix cells add fusogen at 4 0 C 10 min then 37C for 5-10 min; dilute - Total incubation 40 minutes how are membrane proteins visualized? Through tagged secondary antibodies. indirect immunofluorescence immmnofluorescence tagged antibodies indirect = secondary antibodies. incubate with primary antibody that recognizes cell membrane protein incubate with secondary antibody that recognizes the prmary antibody note: secondary antibody is the one that is labeled with fluorescent tag visualize with fluorescence microscopy when are the antibodies added in the experiment? why is this important? After 10 minutes this is important because if we added it before they fused the results would be much different. What is a fluorophore? Is the emission wavelength longer or shorter than excitation wavelength? Why? fluorophore is a is fluroechrome or fluroesecent chromophore. Wavelength is longer because energy conversion is not 100% What is a cell line? What strategies can be used to make one? what happens to the cells? Cell lines are cells that are immortalized and can divide forever. After they reach a certain number of population doubling they reach the hayflick limit meaning the cells will stop dividing. What you can do to have immortal cells is to use the artificial expression of the of the telomerase gene. And maintain at appropriate temperature and gass mixture Understand the general strategy for creating the antibodies Why use two different types of animals (rabbit and mouse) for primary antibody production and only one type of animal (goat) for secondary antibody production? remember difference between polyclonal and monoclonal antibodies, what are the ones being used here? You need two different types of animals to create primary antibodies because we are fusing two different types of cells together, in this case the mouse cell and the human cell. The secondary antibody can be created in the same animal because that animal has the immune system that’s capable of creating a secondary antibody capable of grabbing onto
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the primary antibody. We are using monoclonal antibody, which means that one antibody will stick to one location. How were the antibodies tested to make sure they were specific for human or mouse
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Frye_and_Edidin_highlights_ans - Frye and Edidin highlights...

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