Application of an ectopic expression system for the selection of protein‐isoform‐specific antibodies
- 1 September 1993
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 216 (3), 871-877
- https://doi.org/10.1111/j.1432-1033.1993.tb18209.x
Abstract
Monoclonal antibodies were raised against a fusion protein consisting of a fragment of 141 amino acids of the C-terminal region of the rat brain voltage-gated K(+)-channel protein (RCK1) and the lambda N protein (fusion protein I). Selection of K(+)-channel-specific hybridoma cell lines was performed by means of an ELISA employing a fusion protein consisting of the K(+)-channel-specific peptide sequence and glutathione S-transferase (fusion protein II). For final selection of RCK1 isoform-specific antibodies, a panel of Xenopus oocytes was employed, each injected with cRNA coding for a specific RCK isoform (RCK 1, 2, 4 or 5). Several days after injection, cryosections of embedded oocytes were obtained and were employed in immunohistochemical analysis of antibody binding. Of five hybridoma supernatants from stable growing hybridoma cell lines, selected by the fusion-protein ELISA, one monoclonal antibody (denoted K1C3) recognized exclusively the RCK1-protein isoform, with the other four exhibiting different levels of cross-reactivity with other K(+)-channel isoforms, or with unknown protein(s) of non-injected oocytes. The expression of the RCK1 protein in the postnatal brain was studied using, as far as we are aware, the first example of the application of such isoform-specific antibodies.Keywords
This publication has 30 references indexed in Scilit:
- Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizuresNeuron, 1992
- Differential recognition of a set of O-acetylated gangliosides by monoclonal antibodies RB13-2, D1.1, and JONES during rat brain developmentDevelopmental Brain Research, 1990
- Potassium channels expressed from rat brain cDNA have delayed rectifier propertiesFEBS Letters, 1988
- An emerging pharmacology of peptide toxins targeted against potassium channelsThe Journal of Membrane Biology, 1988
- Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferaseGene, 1988
- The Use ofXenopusOocytes for the Study of Ion ChannelCritical Reviews in Biochemistry, 1987
- Aminoalkylsilane-treated glass slides as support forin situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditionsJournal of Molecular Histology, 1986
- Selective blockage of voltage-dependent K+ channels by a novel scorpion toxinNature, 1982
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970