Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 968-975 |
| Number of pages | 8 |
| Journal | Journal of Lipid Research |
| Volume | 48 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 1 2007 |
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Keywords
- Immunocytochemistry
- Intracellular ceramide localization
- Rabbit immunoglobulin G
ASJC Scopus subject areas
- Biochemistry
- Endocrinology
- Cell Biology
Cite this
Development and characterization of a novel anti-ceramide antibody. / Krishnamurthy, Kannan; Dasgupta, Somsankar; Bieberich, Erhard.
In: Journal of Lipid Research, Vol. 48, No. 4, 01.04.2007, p. 968-975.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development and characterization of a novel anti-ceramide antibody
AU - Krishnamurthy, Kannan
AU - Dasgupta, Somsankar
AU - Bieberich, Erhard
PY - 2007/4/1
Y1 - 2007/4/1
N2 - 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.
AB - 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.
KW - Immunocytochemistry
KW - Intracellular ceramide localization
KW - Rabbit immunoglobulin G
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UR - http://www.scopus.com/inward/citedby.url?scp=34147175939&partnerID=8YFLogxK
U2 - 10.1194/jlr.D600043-JLR200
DO - 10.1194/jlr.D600043-JLR200
M3 - Article
C2 - 17210985
AN - SCOPUS:34147175939
VL - 48
SP - 968
EP - 975
JO - Journal of Lipid Research
JF - Journal of Lipid Research
SN - 0022-2275
IS - 4
ER -