Abstract
Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1,4(trans)-hydroxyproline linkage. Skp1 isoform-specific antibodies were successfully obtained by immunizing mice or rabbits with KLH-coupled synthetic peptides bearing either unmodified Pro, 4(trans)-hydroxyproline, or d-GlcNAcα1,4(trans)-hydroxyproline, and screening with corresponding BSA-conjugates or by Western blotting toward a panel of Skp1 isoforms. Antibodies specific for Skp1 or HO-Skp1 were not found in exhaustive murine trials, yet monospecific polyclonal antibodies were readily achieved in rabbits without cross-adsorption. In all cases, antibodies were specific at the protein but not the peptide level, which suggests that conformation comprises part of the basis for recognition and which should be considered when developing screening strategies.
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Abbreviations
- BSA:
-
Bovine serum albumin
- CRL:
-
Cullin-Ring ligase
- FBP:
-
F-box protein
- KLH:
-
Keyhole limpet hemocyanin
- mAb:
-
Monoclonal antibody
- pAb:
-
Polyclonal antibody
- SCF:
-
Ub-ligase complex consisting of Skp1, cullin-1, an FBP, and Rbx1
- Ub:
-
Ubiquitin
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Acknowledgments
The research summarized here was supported by NIH grants R01-GM037539 and R01-GM084383 and OCAST grant HR-0141.
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West, C.M. et al. (2014). Generating Isoform-Specific Antibodies: Lessons from the Nucleocytoplasmic Glycoprotein Skp1. In: Endo, T., Seeberger, P., Hart, G., Wong, CH., Taniguchi, N. (eds) Glycoscience: Biology and Medicine. Springer, Tokyo. https://doi.org/10.1007/978-4-431-54836-2_170-1
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DOI: https://doi.org/10.1007/978-4-431-54836-2_170-1
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Publisher Name: Springer, Tokyo
Online ISBN: 978-4-431-54836-2
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