PROTEIN INTERACTION SCREENING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Elucidating the functions of the multitudes of newly sequenced proteins, which are the products of the many successful genome projects, is the next major challenge facing biomedical researchers. Complicating this problem, beyond the simple numerical issues, is the fact that many, if not most, proteins are active in large macromolecular complexes. Consequently, it is critical to understand the assembly of these complexes. Flow cytometry offers multiple parameter detection with high sensitivity, excellent kinetic resolution and a homogeneous assay format and these features have been used to study many molecular assemblies in the past. However, such studies have revealed several limitations that prevent flow cytometry from being used to study low affinity molecular assemblies, including poor discrimination of free vs. bound, nonspecific and poor binding of proteins to microspheres, and limited temperature regulation. The purpose of this project is to develop improved methods and instrumentation to enable analysis of and screening for low affinity molecular assemblies. Therefore we will create a flow cytometer utilizing improved excitation optics, sample delivery and data acquisition to enhance instrumental resolution of free vs. bound. We will also evaluate microspheres created of different materials for reduced nonspecific binding and develop improved protein attachment methods. Extending our previously developed Peltier unit technology to the entire sample path of the instrument will enhance temperature regulation. Finally, the above technologies, in conjunction with previous developments, such as the second generation rapid kinetic flow cytometer, will be demonstrated and tested in three collaborative areas, cellular molecular assembly, microsphere based molecular assembly and proteomics in flow. In summary, we will dramatically improve and demonstrate the utility of flow cytometry in the study and screening of low affinity molecular assemblies.