Generation of an antibody against the protein phosphatase 1 inhibitor KEPI and characterization of the epitope.
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 microM. 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.
Daskalow
Katjana
K
Institute of Biochemistry and Biology, University of Potsdam, Golm, Germany.
Boisguerin
Prisca
P
Jandrig
Burkhard
B
Van Landeghem
Frank K H
FK
Volkmer
Rudolf
R
Micheel
Burkhard
B
Schenk
Jörg A
JA
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D054645:Protein Phosphatase 1 / Q000737:chemistry*; D014018:Tissue Distribution
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D047908:Intracellular Signaling Peptides and Proteins; C454464:PPP1R14C protein, human; D010455:Peptides; D054645:Protein Phosphatase 1