Term Papers

859 Words May 22nd, 2015 4 Pages
Section 5 Group 21
Monika Malek: 25%
Megan O’Connor: 25%
Lexy Petrick: 25%
Sophie Sampson: 25%
Experimental Plan
We will explore the relationship between the proteins F58E10.3 and LIN-66, previously identified as a prey and bait, respectively, in a two-hybrid screen. We explore this relationship through a GST pull-down assay, analyzed by gel electrophoresis. If the proteins successfully bind, we expect to see the eluted and bound together GST/bait/prey complex on gel electrophoresis, followed by a final western blot assay. Confirmation of these two proteins interacting is important for controlling developmental timing in C. elegans.

Material and Methods:
MagneGST™ Pull-Down System (purchased from the Promega Catalog),
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* After establishing that GST is being expressed and properly immobilized, we will use mammalian, cell-free expression systems to generate a non-fusion prey protein. We chose to utilize this process as the prey protein will be expressed quickly (a few hours) without needing purification. * To begin this process, we will clone the sequence of the prey protein into a vector containing the appropriate elements for expression using a mammalian-based cell-free expression system. * Establish expression of the prey protein. * Begin the bind/wash/capture cycle. * A cellular extract of cells with the prey expressed will be prepared and allowed to interact with the bound GST bait fusion protein for several hours. * Wash away non-specifically bound proteins. * Elute the bound together GST/bait/prey complex by adding reduced glutathione. * Success of expression and binding will be analyzed by gel electrophoresis, followed by western blot assay, set up as follows (left to right); * A control ladder on the left side of the gel, * lysed E. coli cells expressing the GST tagged bait strain, * a flow-through of lysed bait cells, * a few wash columns (first and second washes), * lysed E.coli cells containing 9XHis-tagged prey protein, * a flow-through of lysed prey cells immobilized with bait, * a few wash columns (first and second

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