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 crossadsorption. 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.
Original language | English |
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Title of host publication | Glycoscience: Biology and Medicine |
Publisher | Springer |
Pages | 927-934 |
Number of pages | 8 |
ISBN (Print) | 9784431548416, 9784431548409 |
DOIs | |
Publication status | Published - 1 Jan 2015 |
Keywords
- Conformational epitope
- Cytoplasmic glycosylation
- Hydroxyproline
- Isoform-specific antibody
- Monoclonal antibody
- Oxygen
- Polyclonal antibody
- Skp1
- Synthetic glycopeptides
- Ubiquitin ligase