title: Isolation of Endoplasmic Reticulum and Its Membrane.
abstract: The association of ribosomes with the rough endoplasmic reticulum (ER) is dependent on Mg2+. The ribosomes can be stripped from the ER by removal of Mg2+from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when ER vesicles are analyzed by equilibrium density gradient centrifugation. Here, I describe the isolation of microsomes from Arabidopsis, followed by the use of the density shift approach in conjunction with equilibrium density gradient centrifugation as a means to diagnose whether a protein is associated with the ER. The same approach can also be used as a means to enrich for ER membranes.
authors.affiliation: Department of Biological Sciences, Dartmouth College, 339 Life Sciences Center, Hanover, NH, 03755, USA. george.e.schaller@dartmouth.edu.
authors.forename: G Eric
authors.initials: GE
authors.lastname: Schaller
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015415:Biomarkers / Q000737:chemistry; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification; D002413:Cations, Divalent; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D025222:Chloroplast Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification; D003470:Culture Media / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008861:Microsomes / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry* / Q000254:growth & development; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification; D012270:Ribosomes / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D015415:Biomarkers; D002352:Carrier Proteins; D002413:Cations, Divalent; D003470:Culture Media; D008565:Membrane Proteins; C077221:BIP protein, Arabidopsis; D013395:Sucrose; D025222:Chloroplast Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D008274:Magnesium
keywords: Arabidopsis; Density shift; Endoplasmic reticulum; Equilibrium density gradient centrifugation; Microsomes; Ribosomes