Generation of plant protein microarrays and investigation of antigen-antibody interactions.
The application of proteomics methods, such as the protein microarray technology, in plant science has been strongly supported by the completion of genome sequencing projects of Arabidopsis thaliana and rice. In this chapter we describe a method to generate plant protein microarrays and to use them for characterizing monoclonal antibodies or polyclonal sera with regard to their specificity and cross-reactivity. The method starts with characterized E. coli cDNA expression clones encoding His-tagged plant proteins. After expression and purification of these recombinant proteins in high throughput, protein microarrays are generated utilizing a contact printer. For the detection of the recombinant proteins on the microarrays, an anti-RGS-His6 antibody is used. To characterize specific antibodies, the microarrays are incubated with the respective antibody solutions followed by fluorescently labeled secondary antibodies. Signal detection is performed by means of an arrayscanner system. Protein microarrays containing the whole proteome of a plant will represent the ideal format to test antibody specificity and cross-reactivity in the future.
Kersten
Birgit
B
RZPD German Resource Center for Genome Research GmbH, Berlin.
Feilner
Tanja
T
D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
D010940:Plant Proteins; D020543:Proteome