Question

You hypothesize that your protein of interest moves from the mitochondria to lysosomes when a cell is exposed to the drug Fantasalin.

A. Design a microscopy experiment to test this hypothesis. Please include the LABELS and type(s) of microscopy you would use. Also, please draw illustrations of what you predict your experiment to look like.

B. Design an experiment to test this hypothesis using cell fractionation and SDS-PAGE. Please include what samples you would collect to run on the gel, and how you will identify your protein of interest. Also please draw an illustration of what you predict your gel to look like.

Answer 1

A.In order to check the hypothesis we can label the protein with GFP i.e Green Fluorescence Protein and then observing it's movement inside live cells before and after treatment with Fantasalin using a Fluorescence microscope. The labelling can be done by inserting the GFP coding sequence at the beginning or end of the coding sequence of the gene encoding our protein of interest. This would result in the production of a fusion protein that can be easily tracked inside live cells due to its GFP tag.

B.Cells can be fractionated by centrifugation of homogenized cell in buffer at progressively higher speeds. Fractions of cells precipitate at different speeds according to their size. Greater the size, lesser the speed required to pellet them down. Hence the different cell fractions obtained by gradually increasing the centrifugation speed and collecting the pellets. These fractions can then be analyzed on SDS PAGE followed by western blot. The fractions that need to be collected to test our hypothesis are mitochondrial fraction and lysosomal fraction before and after treatment with Fantasalin. Our protein of interest can be be identified by first conducting a gel electrophoresis of above mentioned fractions, transfer to nitrocellulose membrane and then treating the membrane with a primary antibody raised against our protein of interest. After washing away the excess primary antibody, the membrane is treated with a secondary antibody that recognizes and binds the primary antibody. The secondary antibody used may be fluoroscently labelled and hence can be detected by immunofluorescence. This would help us detect the presence of our protein of interest in the different fractions isolated. If our hypothesis is true, we would find that our protein of interest is present in mitochondrial fraction and not in lysosomal fraction before addition of Fantasalin and after treatment with Fantasalin, we should find our protein of interest in lysosomal fraction and much decreased amount in mitochondrial fraction.