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Temple University
BIOL 3096
Joel Sheffield

Sheffield Week 9 Monday 10/21/13 Lecture 23 – Research Strategies, Lysosome Radioautography • Following the radio-labeled glucose (pulse) is a whopping dose of non-radio-labeled glucose (chase). This allows the scientist to see the pulse clearly. • The animal's body itself acts as a natural “chase.” But the added non-radio-labeled glucose dilutes the pulse even more. Centrifugation • Insert the radio-labeled glucose, followed by a chase, into the organism, obtain cells, and centrifuge these cells at different speeds to obtain fractions with various organelles. • By doing this with the cells of different animals at progressive time intervals, one can see which organelles the radio-labeled glucose is in at different times, and design a pathway this way. • This is not as precise as radioautography. Using GFP • Introduce a gene for GFP, use microscopy to visualize movement of label. • To reduce amount of fluorescent protein, use a temperature-sensitive mutant • GolgiApparatus is quickly labeled and fluoresces intensely. FRAP- Fluorescence RecoveryAfter Photobleaching • Avariation of the previous technique, an area is photobleached and then the time interval over which fluorescence returns to that area is monitored. • In the example, the GolgiApparatus is photobleached and allows one to clearly see the fluroescent proteins that are traveling to the GolgiApparatus, restoring its fluorescence. 1 | S y d n e s Sheffield Week 9 Monday 10/21/13 Ultimately • These techniques allowed for the development of an image of the pathway of vesicles from the ER to the Golgi to the cell. 1. MRNAtravels out of nucleus, protein is initially
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