Analysis of biomolecular heterogeneity using fluorescence fluctuation spectroscopy

PHYS 2

Richard N. Zare, zare@stanford.edu, Samuel Kim, and Yiqi Luo, rubenluo@stanford.edu. Department of Chemistry, Stanford University, Stanford, CA 94305-5080
We describe the use of a capillary electrophoresis (CE) microchip with a pneumatic valve for separation and collection of fluorescently labeled biomolecules and their complexes. The photon counting histogram (PCH) technique was applied to resolve differently labeled states of the protein sample. The charge difference introduced by the fluorophore itself, which forms a charge ladder, facilitates the separation of the sample. Fluorescence correlation spectroscopy (FCS) was used successfully to identify the separated fractions of immunocomplexes between bovine serum albumin (BSA) and its monoclonal antibodies. Our methodology utilizes both the resolving power of capillary electrophoresis and the PCH and FCS techniques of single-molecule spectroscopy. This approach provides a new way to study heterogeneous populations of biomolecules. Support from the National Science Foundation is gratefully acknowledged.