Immunofluorescence microchamber technique for characterizing isolated organelles.
We describe a rapid technique for the localization and quantitation of specific proteins on organelles bound to microscope chambers. Disposable chambers are constructed from glass slides and provide a platform for the binding of organelles and subsequent immunofluorescence and biochemical assays. Several studies are presented to demonstrate the utility of this technique. Kinesin was visualized in postnuclear supernatants. Golgi and endoplasmic reticulum bound quantitatively to chambers. Endocytic vesicles prepared from rat liver that had been injected in situ with Texas red-labeled asialoorsomucoid allowed for simultaneous detection of asialoorosomucoid, asialoglycoprotein receptor, caveolin 1, and microtubules. Asialoglycoprotein receptor colocalized with asialoorosomucoid-containing vesicles, whereas many of the caveolin 1 structures had no asialoorosomucoid or asialoglycoprotein receptor. The microchambers were also used to measure the binding to endocytic vesicles of exogenously added Rab5 and to monitor the ATP-dependent acidification of endocytic vesicles using the fluorescent dye acridine orange.
Murray
John W
JW
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Bananis
Eustratios
E
Wolkoff
Allan W
AW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D037281:Calnexin; D029968:Escherichia coli Proteins / Q000032:analysis; D019085:Fluorescent Antibody Technique, Direct / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence / Q000379:methods; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000032:analysis
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011506:Proteins; D011956:Receptors, Cell Surface; D014404:Tubulin; D037281:Calnexin; D006180:Proton-Translocating ATPases; D016547:Kinesins