Assignment 1 - Zachary Bergman Cell Bio 1 Based on the...

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Zachary Bergman Cell Bio 1) Based on the methods discussed in this review and in class, propose an experiment to determine whether the DNA for a specific gene associates with the nuclear envelope. The first step should be determining whether our gene interacts with the proteins of the nuclear envelope in an in vitro situation when the two are placed in close proximity to each other. To do this, purify the nucleus and add a GST domain to the proteins of the nuclear envelope. Then run a GST pulldown with the protein and the gene we are studying and if it elutes at the same time as the nuclear envelope proteins it is likely an interaction occurred in which a complex was formed. A possible control for this portion the experiment would be to run a knockout of the nuclear envelope protein in which causes a mutation in the receptor domain and should stop our gene from interacting and thus eluting first. If this is successful we know that they will interact when in close proximity, but not necessarily that they are situated like this in the cell. The next step would be to tag the proteins of the nuclear envelope with a fluorescent tag such as TriTC and image it, then tag our desired gene with another fluorescent tag such as GFP and image it, then merge the images to see if the proteins are localized the same area of the cell (if they are the merged image should have a yellow color at the points of overlap when using these fluorescent tags). A positive control for this would be to tag two nuclear membrane proteins that are known to interact with these same fluorescent markers and determine if the overlapping colors correspond. If both of these tests yield the desired result we have proved that our gene and the nuclear envelope are in the same location and that they interact, at least in the
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Assignment 1 - Zachary Bergman Cell Bio 1 Based on the...

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