Antibodies against CKI1RD, a receiver domain of the sensor
histidine kinase in Arabidopsis thaliana: from antigen
preparation to in planta immunolocalization.

J 2014

Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization.

BORKOVCOVÁ, Petra, Blanka PEKÁROVÁ, Martina VÁLKOVÁ, Radka DOPITOVÁ, Břetislav BRZOBOHATÝ et. al.

Basic information

Original name

Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization.

Authors

BORKOVCOVÁ, Petra (203 Czech Republic, belonging to the institution), Blanka PEKÁROVÁ (203 Czech Republic, belonging to the institution), Martina VÁLKOVÁ (203 Czech Republic, belonging to the institution), Radka DOPITOVÁ (203 Czech Republic, belonging to the institution), Břetislav BRZOBOHATÝ (203 Czech Republic), Lubomír JANDA (203 Czech Republic, belonging to the institution) and Jan HEJÁTKO (203 Czech Republic, guarantor, belonging to the institution)

Edition

Phytochemistry, Elsevier, 2014, 0031-9422

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 2.547

RIV identification code

RIV/00216224:14740/14:00073827

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.1016/j.phytochem.2014.01.007

UT WoS

000333779700001

Keywords in English

CKI1; Immunolocalization; Immunoprecipitation; Polyclonal antibodies; Receiver domain

Abstract

V originále

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.

Links

ED1.1.00/02.0068, research and development project

Name: CEITEC - central european institute of technology

EE2.3.20.0189, research and development project

Name: Rozvoj výzkumné excelence v oblasti evoluční cytogenomiky, epigenetiky a buněčné signalizace

GAP501/11/1150, research and development project

Name: Význam interakcí cytokininů a auxinu v regulaci gravitropismu kořene u Arabidopsis thaliana.

Investor: Czech Science Foundation

GA13-25280S, research and development project

Name: Elucidating molecular mechanisms of cytokinins-ethylene crosstalk in the plant development

Investor: Czech Science Foundation

Citovat

BORKOVCOVÁ, Petra, Blanka PEKÁROVÁ, Martina VÁLKOVÁ, Radka DOPITOVÁ, Břetislav BRZOBOHATÝ, Lubomír JANDA and Jan HEJÁTKO. Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization. Phytochemistry. Elsevier, 2014, vol. 100, April 2014, p. 6-15. ISSN 0031-9422. Available from: https://dx.doi.org/10.1016/j.phytochem.2014.01.007.

@article{1196674,
   author = {Borkovcová, Petra and Pekárová, Blanka and Válková, Martina and Dopitová, Radka and Brzobohatý, Břetislav and Janda, Lubomír and Hejátko, Jan},
   article_number = {April 2014},
   doi = {http://dx.doi.org/10.1016/j.phytochem.2014.01.007},
   keywords = {CKI1; Immunolocalization; Immunoprecipitation; Polyclonal antibodies; Receiver domain},
   language = {eng},
   issn = {0031-9422},
   journal = {Phytochemistry},
   title = {Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization.},
   url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=24529575},
   volume = {100},
   year = {2014}
}

TY  - JOUR
ID  - 1196674
AU  - Borkovcová, Petra - Pekárová, Blanka - Válková, Martina - Dopitová, Radka - Brzobohatý, Břetislav - Janda, Lubomír - Hejátko, Jan
PY  - 2014
TI  - Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization.
JF  - Phytochemistry
VL  - 100
IS  - April 2014
SP  - 6-15
EP  - 6-15
PB  - Elsevier
SN  - 00319422
KW  - CKI1
KW  - Immunolocalization
KW  - Immunoprecipitation
KW  - Polyclonal antibodies
KW  - Receiver domain
UR  - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=24529575
L2  - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=24529575
N2  - 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.
ER  -

BORKOVCOVÁ, Petra, Blanka PEKÁROVÁ, Martina VÁLKOVÁ, Radka DOPITOVÁ, Břetislav BRZOBOHATÝ, Lubomír JANDA and Jan HEJÁTKO. Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization. \textit{Phytochemistry}. Elsevier, 2014, vol.~100, April 2014, p.~6-15. ISSN~0031-9422. Available from: https://dx.doi.org/10.1016/j.phytochem.2014.01.007.

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