Fig. 2. Rab27 purifies with VWF on density gradients. A post-nuclear supernatant was prepared from HUVECs that had been grown to confluence and the organelles contained therein were separated according to density on two sequential self-forming Percoll gradients (aliquots of fractions 7-9 from gradient 1 were pooled and run on gradient 2). (A) The membrane proteins present in each fraction were enriched by TX-114 partitioning, separated according to size by SDS-PAGE and transferred onto a PVDF membrane. The membrane was probed with an antibody to Rab27 and detected with an enzymatically coupled secondary antibody and ECL. (B) The quantification of the Rab27 immunoblot data in (A) (filled black circles) plotted together with the VWF-ELISA values of the same fractions (open squares). Both these data are plotted as percentages of the sum of immunoreactivities across the whole gradient.