Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: Effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

This paper discusses the effects of gel composition and separation temperat ure on the migration properties of fluorescein-5-isothiocyanate-labeled pro tein molecular mass markers (ranging from 20 100 to 205 000 Da) in automate d ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The sep aration mechanism with the agarose and composite agarose - linear polyacryl amide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide m atrices were all found to comply with the Ogston sieving model in the molec ular mass range of the protein molecules investigated. Our temperature stud ies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyeth yl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass o f the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the la ser-induced fluorescent detection of the automated ultrathin-layer electrop horesis system was evaluated by injecting a series of dilutions of the mark ers and was found to be less than 2.5 ng/band for the fluoro-phore-labeled protein.