This chapter describes some simple protocols derived from several earlier methods for obtaining highly purified Golgi stacks from rat liver and for determining their relative purity over the homogenate. Centrifugation is carried out using an L8-70M or Optima LE-80K ultracentrifuge and either a SW-28 rotor containing ultraclear tubes or a SW-41 rotor. It is very important to be as accurate as possible when mixing various components and to check the refractive index of each buffer using a refractometer. Aspirate and discard the lipid at the top and the cytosol and collect Golgi fractions that accumulate at the 0.5/0.86 M interface with a plastic Pasteur pipette. This protocol ensures large amounts of Golgi membranes with minimal contamination by other membranes. The relative purification of the Golgi stacks can be assessed by measuring the increase in specific activity of the Golgi enzyme ?-l,4-galactosyltransferase (GAIT) over that of the whole liver homogenate. The yields of Golgi membranes can be calculated from the ratio between the total GalT activity in the Golgi fractions and that of the homogenate.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)