Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

The overarching goal of this procedure is to identify protein, protein interactions within a given proteome in a parallel unbiased manner, which is not possible with other technologies. This is accomplished by first hybridizing and epitope tagged protein of interest with a highly purified, full length functional protein library deposited in a coordinate plane on the surface of a microscope slide. Once the protein has reached equilibrium with each of its respective target proteins on the array, the unbound fraction is washed away and the protein interactions are detected with a monoclonal antibody to the epitope conjugated to a fluorescent moiety using a standard microarray scanner.

The resulting images are interlaced with information regarding the intensity of each feature with respect to bound protein and fluorescent signal. This information is extrapolated with the aid of the microarray analysis software to generate a list of intensity values for each feature on the array. The information regarding the protein-protein interacting pair can be exported to downstream bioinformatic analysis software to score the feature specific data and identify protein protein interactions between the protein of interest and the proteins on the microarray.