Preparative agarose gel electrophoresis for the purification of small organelles and particles

Abstract

A novel preparative method of quantitative flatbed agarose gel electrophoresis has been used to separate a number of small subcellular structures, such as ribosomes, coated vesicles, smooth vesicles, and ferritin. The technique utilizes continuous elution of a second, electrophoretically “downstream”, well in the agarose gel. The elution occurs concurrently with the electrophoresis, so essentially no additional time is required for the recovery of the structures. The technique is nondestructive, relatively simple and inexpensive, and can be used by modifying any nonsubmerged horizontal agarose gel system. The preparative separation of small organelles and subcellula structures according to their charge allows the purification of small structures previously difficult to isolate by conventional techniques. Two novel structures purified by this technique are described: a short intermediate filament-like species consisting of a single polypeptide of M r 142,000, and an ovoid species (70 × 35 nm) whose protein composition is dominated by a polypeptide of M r 104,000.