A novel lectin-gold density perturbation eliminates plasma membrane contaminants from Golgi-enriched subcellular fractions.
The availability of pure Golgi fractions is a prerequisite for documenting the composition of the membranes of the Golgi Complex and comparing and contrasting this organelle with the rough endoplasmic reticulum. In a companion article, we have described a subcellular fractionation protocol for rat myeloma cells which effectively eliminates rough microsomes from Golgi-enriched fractions. Nevertheless, a major overlap with plasma membrane remains. We have therefore developed a novel density perturbation procedure to eliminate plasma membrane contaminants. By binding a conjugate of wheat germ agglutinin and colloidal gold to cells at 4 degrees C before homogenization we cause extensive sedimentation of plasma membrane markers to the "mitochondrial pellet" as well as a major shift in the isopycnic density of these markers. The differential and isopycnic sedimentation of several Golgi markers is unaffected in lectin-gold treated cells. The Golgi-enriched fractions obtained by isopycnic sedimentation are therefore of high purity. This procedure may be of general use for either purifying or eliminating plasma membrane-derived vesicles. Adaptations of the method might be equally useful for density perturbation of intracellular organelles.
Gupta
D K
DK
Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106.
Tartakoff
A M
AM
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure; D006046:Gold / Q000032:analysis / Q000378:metabolism*; D006056:Golgi Apparatus / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure; D037102:Lectins / Q000032:analysis / Q000378:metabolism*; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D051381:Rats; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D037102:Lectins; D014909:Wheat Germ Agglutinins; D006046:Gold