Term Papers

866 words - 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™ ...view middle of the document...

* Immobilize the GST bait fusion protein on a support resin using glutathione particles. These particles are optimal because they bind to the GST tag.
* 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 washes),
* eluted bait protein with binding buffer,
* eluted prey protein with binding buffer,
* eluted bait-prey complex from agarose beads containing 100mM reduced glutathione.

Expected Results:
If there is indeed interaction between a prey extracted from C. elegans...

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