Submitted on October 27, 2006
Accepted on January 8, 2007
Development and characterization of a novel anti-ceramide antibody
Kannan Krishnamurthy, Somsankar Dasgupta, and Erhard Bieberich
Medical College of Georgia, Augusta
Corresponding Author: ebieberich{at}mail.mcg.edu
Ceramide is emerging as a key sphingolipid that regulates a variety of cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins we have developed a novel antibody against ceramide. Our results indicate that the antibody (rabbit IgG) specifically recognizes ceramide in lipid overlay assays and detects ceramide species with different fatty acid chain lengths that include C2, C8, C16, C18, C20 and C24. The new antibody was compared with the commercially available anti-ceramide antibody (mouse IgM) in immunocytochemistry experiments to study the localization of ceramide. While both antibodies stain the same regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in compartments that are not well identified with the commercially available antibody. In addition to staining of ceramide in protrusions of the plasma membrane, the rabbit IgG also detects ceramide in the Golgi apparatus. Pharmacological depletion or elevation of ceramide levels results in a corresponding change in staining intensity, thus confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine localization of ceramide and its interaction with proteins by immunocytochemistry.
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