Question

You want to study the timing of the APC's activity more distinctly. To do this you treat your cells with MG132 - a proteasome inhibitor. When MG132 is added to cells proteins can still be ubiquitinated, but they are not degraded, because the proteasome is inhibited (4pts) In cells that are treated with MG132 you can monitor APC activity on Cyclin B using just the cyclin B a. antibody and a western blot, and monitoring the size of cyclin B. Describe how and why this works.

Answer 1

Ans. The various stages of mitosis are prophase, metaphase, anaphase and telophase. The transition from one stage to another is set up by degradation of previous cyclin proteins and formation of new protein complexes. The term APC stands for Anaphase Promoting Complex. This complex set of proteins initiates the onset of anaphase in a cell undergoing division.

The protein Cyclin B is seen to be related to the onset of anaphase. Degradation of this protein leads to transition from metaphase to anaphase. In the given case, we want to study the timing of APC activity. For this MG132 is added to the cell. Now, this proteasome can ubiquinate the proteins, but it cannot degrade them. So, according to the problem Cyclin B proteins have been ubiquinated, but as they are not degraded so they will delay the cell from entering anaphase by forming Anaphase Promoting Complex.

a. Next, when cyclin B antibody will be added to these cells, it will lead to binding of these antibodies with Cyclin B proteins and their degradation. In order to identify specific proteins, Western blot technique is used. In this method, specific proteins are detected from a mixture of proteins by binding these proteins with their specific antibodies. This marks the specific proteins and then they are separated by using gel electrophoresis method.

So, in the given case when Western blot technique is performed, we see a clear band of APC complex and cyclin B antibody bound to Cyclin B protein, so the size of this antibody increases. As the APC complex becomes active, it degrades any more Cyclin B present in the cell. This leads to a faint band of cyclin B antibody-Cyclin B protein with time and a prominent band of only cyclin B antibody is seen by the end of anaphse.