Gel electrofocusing in tubes containing Sephadex

Abstract

AbstractGel electrofocusing (EF) on Sephadex G‐75, which provides relatively non‐restrictive pore sizes and relatively broad protein concentration distributions (which reduces concentration‐dependent aggregation), was adapted for use in glass tubes. This allows for EF with meticulously controlled low temperatures in conjunction with large sample and gel volumes. Specifically, the technique was developed for EF of steroid receptors, which are highly sensitive to temperature (temperature transformed), and whose aggregation behavior excluded the use of polyacrylamide or agarose gels. The technique uses the temperature‐controlled apparatus designed for quantitative polyacrylamide gel electrophoresis, with three additions. These are a) a 12‐place evacuation device for forming simultaneously 12 cylindrical Sephadex gels (either 6 or 18 mm diameter), b) an electrode arrangement capable of maintaining the same regulated voltage across several gel tubes, and c) a jacket and lid of polyurethane foam to insulate the electrolyte chambers of the EF apparatus.