Measuring Protein Movement, Oligomerization State, and Protein-Protein Interaction in Arabidopsis Roots Using Scanning Fluorescence Correlation Spectroscopy (Scanning FCS).
Scanning fluorescence correlation spectroscopy (scanning FCS) can be used to determine protein movement, oligomerization state, and protein-protein interaction. Here, we describe how to use the scanning FCS techniques of raster image correlation spectroscopy (RICS) and pair correlation function (pCF) to determine the rate and direction of protein movement. In addition, we detail how number and brightness (N&B) and cross-correlation analyses can be used to determine oligomerization state and binding ratios of protein complexes. We specifically describe how to acquire suitable images for scanning FCS analysis using the model plant Arabidopsis and how to perform the various analyses using the SimFCS software.