Reproducibility of mobility in gel electrophoresis

The quantitative exploitation of gel electrophoresis to yield molecular and gel fiber properties rests on the assumption that mobility is characteristic of the macromolecule migrating as a band and is a physical constant for any system defined by pH, ionic strength and temperature. This assumption has not been tested intra-experimentally in previous literature. With the commercial introduction of automated gel electrophoresis apparatus, the collection of multiple mobility data during a single run without additional expense of labor has made it possible to test the assumption. As a start, we undertook that test for three proteins and their sodium dodecyl sulfate (SDS) derivatives, in agarose and polyacrylamide gel electrophoresis, various field strengths, continuous and discontinuous buffers, as well as intra- and interexperimentally. It was found that in agarose gel electrophoresis conducted in a single buffer, the standard deviation of mobility over a wide concentration range ranges intra-experimentally from 0.2 to 1.3% for two globular proteins and 1.4 to 5.3% for the same proteins derivatized with SDS. Interexperimentally, it was 3% in the single case tested to date. The standard deviation in polyacrylamide appears to be higher, varies in inverse relation to the mobility value, i.e. increases with gel concentration in the range of 11 to 19%T, and varies substantially between the two SDS-proteins investigated. Mobility in a discontinuous buffer system decreases continuously due to the decreasing leading phase/trailing phase ratio along the migration path. The decrease is sharpest in the "nonrestrictive" stacking gel.