title: Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization.
abstract: Immunodetection is a powerful tool in functional studies of all organisms. In plants, the gene redundancy and presence of gene families composed of highly homologous members often impedes the unambiguous identification of individual gene products. A family of eight sensor histidine kinases (HKs) mediates the transduction of diverse signals into Arabidopsis thaliana cells, thereby ensuring the initiation of appropriate adaptive responses. Antibodies recognizing specific members of the HK family would be valuable for studying their functions in Arabidopsis and other plant species including important crops. We have focused on developing and applying antibodies against CYTOKININ-INDEPENDENT 1 (CKI1), which encodes a constitutively active membrane-bound sensor HK that regulates the development of female gametophytes and vascular tissue in Arabidopsis. A coding sequence delimiting the C-terminal receiver domain of CKI1 (CKI1(RD)) was expressed in Escherichia coli using the IPTG-inducible expression system and purified to give a highly pure target protein. The purified CKI1(RD) protein was then used as an antigen for anti-CKI1(RD) antibody production. The resulting polyclonal antibodies had a detection limit of 10 ng of target protein at 1:20,000 dilution and were able to specifically distinguish CKI1, both in vitro and in situ, even in a direct comparison with highly homologous members of the same HK family AHK4, CKI2 and ETR1. Finally, anti-CKI1(RD) antibodies were able to selectively bind CKI1-GFP fusion protein in a pull-down assay using crude lysate from an Arabidopsis cell suspension culture. Our results suggest that the receiver domain is a useful target for the functional characterization of sensor HKs in immunological and biochemical studies.
authors: Functional Genomics and Proteomics of Plants, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5/A2, CZ-625 00 Brno, Czech Republic.
authors: Petra
authors: P
authors: Borkovcová
authors: Functional Genomics and Proteomics of Plants, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5/A2, CZ-625 00 Brno, Czech Republic.
authors: Blanka
authors: B
authors: Pekárová
authors: Functional Genomics and Proteomics of Plants, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5/A2, CZ-625 00 Brno, Czech Republic.
authors: Martina
authors: M
authors: Válková
authors: Functional Genomics and Proteomics of Plants, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5/A2, CZ-625 00 Brno, Czech Republic.
authors: Radka
authors: R
authors: Dopitová
authors: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, CZ-612 65 Brno, Czech Republic; Department of Molecular Biology and Radiobiology, CEITEC - Central European Institute of Technology, Mendel University of Agriculture and Forestry, Zemědělská 1, CZ-613 00 Brno, Czech Republic.
authors: Břetislav
authors: B
authors: Brzobohatý
authors: Functional Genomics and Proteomics of Plants, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5/A2, CZ-625 00 Brno, Czech Republic.
authors: Lubomír
authors: L
authors: Janda
authors: Functional Genomics and Proteomics of Plants, CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5/A2, CZ-625 00 Brno, Czech Republic. Electronic address: hejatko@sci.muni.cz.
authors: Jan
authors: J
authors: Hejátko
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015398:Signal Transduction
chemical_list: D000906:Antibodies; D029681:Arabidopsis Proteins; D011494:Protein Kinases; C101990:CKI1 protein, Arabidopsis
keywords: CKI1; Immunolocalization; Immunoprecipitation; Polyclonal antibodies; Receiver domain