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    Protein Expression and Pulse Chase Analysis

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    I want to study a human protein(protein X) that is normally secreted from the cell. I express the gene for Protein X in cultured Rat cells. I conduct a pulse-chase experiment in which I specifically label Protein X(and no other cellular protein) at 37 degrees celcius and then perform a 35 minute chase also at 37 degrees celcius. I follow protein X's intracellular distribution during subcellular fractionation on a density gradient. Panel (a) shows the result immediatley after the pulse. Panel (b) shows the result after 35 minutes of chase.

    I dont understand what is happening going from panel A to panel B
    I want to know how panel (b) would look if I attacheed a KDEL sequence to the gene for protein X.

    How will Panel (b) would look if both the pulse and chase were conducted on ice (2-4 degrees celcius)

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    Solution Preview

    Panel A clearly shows that the protein is made on ribosomes bound at the ER. How do we know this? Because the radioactivity is found exclusively at the ER. If protein X was made in the cytosol, we wouldn't see the radioactivity at the ER, right?

    Now, Panel B shows that after a while, that most of the radioactivity has moved onto the ...

    Solution Summary

    Protein expressions and pulse chase analysis are examined. The subcellular fractionation on a density gradients are provided.