Separation of Proteins by Ammonium Sulfate Gradient Solubilization

Abstract

The classical method of protein separation by ammonium sulfate precipitation can be carried out in the reverse manner. Namely, the proteins are first precipitated in the presence of a carrier and then they are solubilized with a decreasing salt gradient. An advantage of the reverse procedure is that it makes it possible for one to choose the best recovery of the desired protein with the least amount of contaminants. The reverse procedure has been reported by other workers but the various parameters involved had not been studied. The present results show that the width of a solubilized protein peak covers a 5-7 % saturation range of ammonium sulfate for 85 % recovery of the sample, and that the salt saturation at the protein peak depends not only on the nature of the protein but also on its amount and the gradient used. With a given gradient there is an optimum as to the amount of proteins which may be separated; too large an amount will give a decreased resolution of the peaks and too small an amount will give poor recovery. The simplicity of the solubilization procedure facilitates investigations of the influences pH and temperature on the desired separation. The usefulness of this procedure is illustrated with the isolation of the two major allergens from ragweed pollen, antigens E and K. © 1972, American Chemical Society. All rights reserved.