Question 1: Explain how map kinase is phosphorylated in response to NGF stimulation (maximum 200 words) Question 2: Explain the principles of how Western blotting works. (no maximum on each of these sections but each section should be able to be answered in a couple of sentences) I. Why are the proteins in the PC 12 cell extracts incubated with ß-mercaptoethanol, sodium dodecyl sulphate (SDS) and then heated to 100°C II. What is the basis of the separation of proteins by SDS gel electrophoresis III. Why is the gel incubated with transfer buffer prior to electrophoretic transfer IV. Why do we transfer the proteins to the solid support, the nitrocellulose filter? Why can’t the antibody detection of map kinase be done when it is still in the gel? V. After the proteins have been transferred to the nitrocellulose membrane why is the nitrocellulose membrane incubated in blocking buffer prior to addition of the two different primary antibodies? VI. What form/s of map kinase does the anti-total map kinase antibody detect? VII. What form/s of map kinase does the anti-active map kinase antibody detect? VIII. Why does an intense purple colour develop on the nitrocellulose membrane at the position of the map kinase molecules? Question 3: What do the results of the experiment that are presented by Sam at the end of the video tell you about the effect of NGF stimulation of PC12 cells on the level of total map kinase and the level of map kinase that is phosphorylated and active? (maximum 200 words) Question 4: Why are there map kinase proteins with different molecular weight? (maximum 200 words)

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