Isolation of peroxisome subpopulations from rat liver by means of immune free-flow electrophoresis

Immune free-flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen-specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pi of IgG molecules (pH similar to 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG-coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post-mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.