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Originally published In Press as doi:10.1194/jlr.D600043-JLR200 on January 8, 2007

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Journal of Lipid Research, Vol. 48, 968-975, April 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Development and characterization of a novel anti-ceramide antibody

Kannan Krishnamurthy, Somsankar Dasgupta and Erhard Bieberich1

School of Medicine, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912

Published, JLR Papers in Press, January 8, 2007.

1 To whom correspondence should be addressed. e-mail: 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. Although 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 increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine the localization of ceramide and its interaction with proteins by immunocytochemistry.

Supplementary key words rabbit immunoglobulin G • intracellular ceramide localization • immunocytochemistry

Abbreviations: GlcCer, glucosylceramide; KLH, keyhole limpet hemocyanin; NB-dNJ, N-butyl-deoxynojirimycin


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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.