Generation of an antibody against the protein phosphatase 1 inhibitor KEPI and characterization of the epitope

Item Type:	Article
Title:	Generation of an antibody against the protein phosphatase 1 inhibitor KEPI and characterization of the epitope
Creators Name:	Daskalow, K. and Boisguerin, P. and Jandrig, B. and Van Landeghem, F.K. and Volkmer, R. and Micheel, B. and Schenk, J.A.
Abstract:	A monoclonal antibody against the potential tumor suppressor kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI (PPP1R14C) was generated and characterized. Human KEPI was expressed in Escherichia coli and used to immunize Balb/c mice. Using hybridoma technology, one clone, G18AF8, was isolated producing antibodies which bound specifically to the KEPI protein in ELISA, immunoblotting and flow cytometry. The antibody was also successfully applied to stain KEPI protein in paraffin sections of human brain. The epitope was mapped using peptide array technology and confirmed as GARVFFQSPR. This corresponds to the N-terminal region of KEPI. Amino acid substitution analysis revealed that two residues, F and Q, are essential for binding. Affinity of binding was determined by competitive ELISA as 1 muM. In Western blot assays testing G18AF8 antibody on brain samples of several species, reactivity with hamster, rat and chicken samples was found, suggesting a broad homology of this KEPI epitope in vertebrates. This antibody could be used in expression studies at the protein level e.g. in tumor tissues.
Keywords:	Hybridoma, Monoclonal, KEPI, Peptide Array, Substitutional Analysis, Animals, Mice
Source:	Anticancer Research
ISSN:	0250-7005
Publisher:	Delinassios
Volume:	30
Number:	5
Page Range:	1573-1578
Date:	May 2010
Official Publication:	http://ar.iiarjournals.org/content/30/5/1573.abstract
PubMed:	View item in PubMed

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