Estrogen receptor separation employing sucrose deuterium oxide gradients generated during sample centrifugation

Abstract

A quick and easy method of sucrose density gradient analysis is described in which prior preparation of gradients for protein separation is not required. We compare the refractive indices as well as the separation pattern of radioactively labeled proteins with either preformed gradients (prepared by layering or by gradient maker) or gradients that establish themselves during centrifugation. Our results show that an identical pattern of protein separation can be achieved by layering the nuclear extracted estrogen receptor either on top of one single sucrose concentration or on a preformed gradient. Centrifugation times are 40 hr in a swinging bucket type rotor (Sw₆₀) or 15 hr in a vertical tube rotor (VTi 65). This method avoids the onerous task of gradient preparation, and the separation itself can be achieved in a 15 hr centrifugation by use of a vertical tube rotor.