(a) Setup for a pulse-chase experiment Before experiment Pulse Chase The cartoon
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(a) Setup for a pulse-chase experiment Before experiment Pulse Chase The cartoon of the cells to the right represents what Palade observed. The results are quantified in (b). Rough ER What do you see in the graph? Golgi apparatus Secretory vesicles Labeled What can you conclude? proteins (b) Tracking pulse-labeled proteins during the chase You should be able to clearly explain how a pulse chase experiment tells you how a protein's transport through a cell occurs 90 801 2 70 a 60 Rough ER Golgi apparatus 40 30- 20 10- vesicles 07 17 37 57 Incubation time after pulse (min)Explanation / Answer
1)graph shows the time course of movement of a labelled protein through compartments in a secretory cell. This type of information can be obtained by cell fractionation .
2)In this procedure cells are gives a short exposure to a high concentration of radioactively labelled material.This is the pulse part of the experiment. This is then followed by removal of the labelled medium and the administration of a great excess of non-labelled material. This addition of non-labelled amino acid greatly reduces the specific activity of the leucine in the cell. This is the chase part of the experiment.labelled molecules were made during the few minutes that the high activity label was present in the system.
3)Cell fractionation is used to follow labelled molecules in this pulse chase experiment.population is sampled at different times after the start of the chase (e.g. 3 min, 5 min, 10, 15, 20, 30, 60 min), then lyse the cells and separate the bits of ER, Golgi, Secretory granules, extracellular material etc by differential centrifugation and other biochemical procedures and determine how much time elapses before label appears in secretory granules .
4)graph shows the time course of a labelled protein as it is processes for the secretory process. In this experiment acinar cells of the pancreas were given a labelled amino acid which was incorporated into proteins during the first minutes of the experiment. The labelled amino acid was then replaced by a large excess of non-labelled amino acid (chased), the the labelled material already incorporated into proteins was followed through the secretory process.
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