FIG. 3.
Production and purification of SCoV VLPs. Cultures of 293T cells were transfected with 14 μg of pCAGGS-S, 1.4 μg of pCAGGS-M, 2.8 μg of pCAGGS-N, and 9 μg of pCAGGS-E. The cells were labeled with Tran[35S] for 6 h from 42 to 48 h posttransfection. The culture media were clarified, and VLPs were purified by centrifugation on a discontinuous sucrose gradient and on a subsequent continuous sucrose gradient. Ten fractions were collected from the bottom of the gradient. VLP samples in each fraction were pelleted by centrifugation through 20% sucrose cushions. The pellets were dissolved in 1× SDS-PAGE loading buffer and analyzed using SDS-PAGE and autoradiography.