High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions.
We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.
Tulp
A
A
The Netherlands Cancer Institute, Amsterdam. atulp@nki.nl
Fernandez-Borja
M
M
Verwoerd
D
D
Neefjes
J
J
D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification; D002256:Carbonic Anhydrases / Q000302:isolation & purification; D002417:Cattle; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes; D006801:Humans; D007525:Isoelectric Focusing; D007782:Lactoglobulins / Q000302:isolation & purification; D015388:Organelles; D011506:Proteins / Q000302:isolation & purification*; D013347:Subcellular Fractions; D014168:Transferrin / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D001059:Apoproteins; D007782:Lactoglobulins; D011506:Proteins; D014168:Transferrin; C028284:apotransferrin; D002256:Carbonic Anhydrases