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Monoclonal antibodies against rat liver mitochondrial phospholipase A2: epitope analysis and application in western
R H van Schaik1, C D van den Koeduk, F W Neijs
1Center for Biomembranes and Lipid Enzymology, University Utrecht, The Netherlands.
1. Eight cell-lines producing monoclonal antibodies, raised against rat liver mitochondrial phospholipase A2, were investigated with respect to epitope-recognition. It was shown that all antibodies tested were directed to an identical epitope. 2. This epitope is a conformational one, since treatment of phospholipase A2 with the reducing agent dithiothreitol lowered the antibody binding significantly. 3. To increase sensitivity, Western blot analyses have to be performed on protein samples lacking dithiothreitol or beta-mercaptoethanol. The conditions described in this report allow the detection of the phospholipase A2 in rat liver homogenates. 4. When rat liver mitochondrial phospholipase A2 was purified by Ultrogel AcA 54 gel filtration, a nearly homogeneous protein preparation was obtained, as judged by SDS-PAGE. Western blot analysis of this preparation, however, clearly indicated the phospholipase A2 to correspond to a hardly visible protein band.
1. Eight cell-lines producing monoclonal antibodies, raised against rat liver mitochondrial phospholipase A2, were investigated with respect to epitope-recognition. It was shown that all antibodies tested were directed to an identical epitope. 2. This epitope is a conformational one, since treatment of phospholipase A2 with the reducing agent dithiothreitol lowered the antibody binding significantly. 3. To increase sensitivity, Western blot analyses have to be performed on protein samples lacking dithiothreitol or beta-mercaptoethanol. The conditions described in this report allow the detection of the phospholipase A2 in rat liver homogenates. 4. When rat liver mitochondrial phospholipase A2 was purified by Ultrogel AcA 54 gel filtration, a nearly homogeneous protein preparation was obtained, as judged by SDS-PAGE. Western blot analysis of this preparation, however, clearly indicated the phospholipase A2 to correspond to a hardly visible protein band.