Removal of percoll from microsomal vesicles by gel filtration on sephacryl-S-1000 superfine.
A microsomal vesicle fraction was prepared from rat liver homogenate by centrifugation in gradients of Percoll. The microsomes were subjected to gel filtration on Sephacryl S-1000 Superfine, which resolved the microsomes from Percoll. The elution pattern of the microsomal marker enzyme NADPH-cytochrome c reductase showed that the main part of the enzyme was present in a peak at Kav about 0.1, while Percoll eluted in a broad peak at Kav about 0.7. The total yield of eluted enzyme activity was 85%. The gel filtration had to be carried out in the presence of 10 mM tris or NaCl. At lower ionic strength or in 0.25 M sucrose alone, anomalous behaviour of the Percoll particles and microsomes on the gel was observed. Electron microscopy of samples from the void volume fraction of the Sephacryl S-1000 Superfine column showed an almost complete removal of Percoll from the microsomes. Furthermore, the vesicle preparation was essentially free of membrane fragments.
D000180:Acrylic Resins; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D008099:Liver / Q000648:ultrastructure; D008297:Male; D008861:Microsomes; D011205:Povidone / Q000302:isolation & purification*; D051381:Rats; D012822:Silicon Dioxide / Q000302:isolation & purification*
D000180:Acrylic Resins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone