We have developed real-time full-field fluorescence polarization microscopy to monitor the dynamic fluorescence anisotropy from the cationic tetramethy-lindocarbocyanine (DiI) dyes during SDS-PAGE. Cationic dyes (e.g., DiI) localize in the "extended stack" (ES), a concentrated detergent zone directly behind the moving boundary below 15%T. We will present quantitative results of the fluorescence anisotropy in the ES zone.

The moving boundary [or Leading/Trailing boundary (LT)] was detected in real time with the addition of pH indicator dyes to the gel matrix. Pre-cast commercial Tris-Tricine-Acetate 8-25%T gels were run in a cross-gradient fashion with the addition of pH dyes to demonstrate a distinct pH gradient at the moving boundary only in the presence of the dodecyl sulfate. The pH gradient at the moving boundary is dependant both on the presence of the concentrated dodecyl sulfate (ES) zone in the gel and %T of the gel.

We have proposed a model linking the fluorescence anisotropy in the extended stack and pH data at the moving boundary. It suggests that increased detergent structure beyond the current free-solution micelle model plays an important role in relative protein mobility near the moving boundary. Sequential digital images of the fluorescence anisotropy and pH dye data will be displayed.

Typically the mobility of SDS-coated proteins varies logarithmically with total acrylamide concentration (%T). We have previously shown that changes in pH at constant %T also show a logarithmic dependence from pH 6.8 to 9.8 using a Tris-Glycine-Chloride method (after Laemmli).

Evidence for improved low molecular weight (5-50 kDa) separation independent of %T in SDS-PAGE will be shown. Since the anionic detergent concentration in a gel is at least 1010 greater than the concentration of protein molecules these studies will help us to obtain a better understanding of the real-time detergent properties throughout the process of SDS-PAGE.