Development and characterization of a novel anti-ceramide antibody
Development and characterization of a novel anti-ceramide antibody
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DOI：
10.1194/jlr.d600043-jlr200
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发表时间：
2007-04-01
影响因子：
6.5
通讯作者：
Bieberich, Erhard
中科院分区：
文献类型：
--
作者：
Krishnamurthy, Karman;Dasgupta, Somsankar;Bieberich, Erhard
神经酰胺是一种重要的鞘脂，调节各种细胞过程。为了便于研究神经酰胺的定位及其与细胞蛋白质的相互作用，我们开发了一种新的抗神经酰胺抗体。我们的研究结果表明，抗体（兔IgG）特异性识别神经酰胺在脂质覆盖试验和检测神经酰胺种类与不同的脂肪酸链长度，包括C2，C8，C16，C18，C20和C24。在免疫细胞化学实验中，将新抗体与市售的抗神经酰胺抗体（小鼠IgM）进行比较，以研究神经酰胺的定位。尽管两种抗体染色细胞膜上的相同区域，但兔IgG显示神经酰胺在用市售抗体不能很好鉴定的隔室中的分布。除了质膜突起中的神经酰胺染色外，兔IgG还检测高尔基体中的神经酰胺。神经酰胺水平的药理学消耗或增加会导致染色强度发生相应的变化，从而证实抗体的特异性。这些结果表明，兔IgG是一个合适的抗体，以确定神经酰胺的定位及其与蛋白质的相互作用的免疫细胞化学。
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.