Detection of bis(sulfosuccinimidyl) suberate binding in electrophoresis: determination of membrane sidedness of proteins |
| |
Authors: | Till Olaf Schmidt Martin Linss Werner Baumann Eckehard |
| |
Affiliation: | Institute of Anatomy I, Hospital of the Friedrich Schiller University, Jena, Germany. olaf.till@uni-jena.de |
| |
Abstract: | The applicability of the membrane-impermeant protein cross-linker bis(sulfosuccinimidyl) suberate (BS(3)) to the determination of membrane sidedness of proteins was tested in 3T3-L1 cells and in erythrocytes. Binding of BS(3) to proteins was apparent in electrophoresis. In three proteins of 3T3-L1 cells, protein kinase-Cepsilon, protein kinase-Czeta, and glyceraldehyde-3-phosphate dehydrogenase, BS(3) action was detectable in SDS-PAGE with immunoblotting. This enabled confirmation of the well-known intracellular localization of these proteins. In cathepsin E of erythrocytes, a mobility increase in nondenaturing PAGE was the most prominent effect of BS(3) treatment. A mechanism for the increase in mobility due to BS(3) binding is suggested. Cathepsin E was found to be located at the intracellular side of the membrane, in accordance with existing evidence. |
| |
Keywords: | Bis(sulfosuccinimidyl) suberate Cross‐linking Membrane topology Nondenaturing PAGE SDS‐PAGE |
本文献已被 PubMed 等数据库收录! |
|