Aaron V. Riley 
Marquette University
Milwaukee, WI
Faculty Mentor: Dr. Gail L. Waring

The Drosophila eggshell is a complex, specialized extracellular matrix that contains three morphologically distinct layers made of protein. The locations of some of these proteins are known. For example, the current protein of interest, s25, is found in all three layers. However, no one has yet determined any protein-protein interactions between the eggshell proteins. To begin to investigate these interactions, a technique known as a Far Western Blot will be used. In a Far Western, instead of probing a protein blot with an antibody, one probes with another protein, taking advantage of specific protein-protein interactions.

To obtain this protein probe, a desired gene region will be cloned into a bacterial expression vector containing a hexahistidine tag and a site for in vitro phosphorylation. After protein expression, the protein will be purified by affinity chromatography taking advantage of the His-tag and its affinity for Ni-chelate columns. Once the protein is purified, the site that allows for phosphorylation will be labeled with ³²P. The labeled protein will then be used to probe blots of egg chamber extracts from different developmental stages.

Once potential interacting partners are identified, the molecular motifs necessary for the interactions can be determined by selectively mutating the protein probes. To test whether the interacting motif is required in vivo, mutated versions of the probe protein (e.g. s25) can be introduced into flies and its effects on eggshell assembly can be followed. By identifying molecular motifs important in eggshell assembly, insight will hopefully be gained not only on the assembly of the Drosophila eggshell, but other extracellular assembly processes as well.