Charge determination of membrane molecules in polymer-supported lipid layers.

A method for two-dimensional micro-electrophoresis and charge determination of fluorescence-labeled membrane molecules in lipid layers is presented. Therefore, the labeled molecules are dissolved in a lipid monolayer which acts as a fluid matrix. The essential part of the sample preparation is an aqueous polymer film composed of agarose onto which the layer is transferred by Langmuir-Blodgett technique. The induced force of an applied electric field leads to a drift of the charged fluorescent molecules. The mobility is determined by an analysis of the steady-state bleach profile which is produced by continuous fluorescence micro-photolysis of a rectangular area of the monolayer. Testing a variety of amphiphilic molecules, measurements yielded values of zero, plus or minus one elementary net charge within a margin of error. The experimental set-up described here can be used for lateral separation, enrichment and isoelectric focusing of membrane components.