Tanja Feilner's research while affiliated with University College Cork and other places

... Using this technique to investigate A. thaliana transcription factor (TF) interactions, a deep-coverage A. thaliana TF interaction network was created (AtTFIN-1), greatly expanding the number of known plant TF interactions ( Trigg et al., 2017). Other systematic approaches include classic in vitro protein arrays (Feilner et al., 2005;Popescu et al., 2007Popescu et al., , 2009 • Interactome studies mentioning hubs are marked with a gray background. ...

... Conventional detection methods for microarrays often use fluorescent dyes, which were detected by irradiation from a laser scanner. Detection methods are discussed intensively elsewhere (Espina et al. 2004;Feilner et al. 2004), while in this study, only new approaches should be pointed out to reach much higher sensitivities. With a special method called MIST (multiple spotting technique) (Angenendt et al. 2003a, b), multiple spotting steps are performed successively to one position. ...

... Proteomics is one of the high-throughput approaches currently being used to address biological function of plant by studying globally expressed proteins in a given tissue. [2][3][4][5][6][7][8][9][10] Two-dimensional gel electrophoresis (2-DGE) is the most commonly used proteomics technology for monitoring changes in the expression levels of complex protein mixtures, and is also the most widely utilized. 2,8-10 2-DGE can simultaneously separate thousands of proteins (and their modified forms) to homogeneity, deliver high-quality protein resolution and dynamic range, and generate reference maps in a short period of time, and at a cost "affordable" to proteomics researcher. ...

... Therefore, protein microarrays were also employed as a tool for phosphoproteomic analysis, as this allows detection of phosphorylation targets as well as molecular interactions using thousands of immobilized probable protein targets [32][33][34][35]. This technique was first employed for A. thaliana proteins [36]. Before designing the high-throughput microarrays with A. thaliana kinases [37], low throughput microarrays were designed to understand the number of microarrays analyzed for one phosphorylation on one microarray [38,39] followed by medium throughput with barley kinases [40]. ...

... To quantify specific proteins, antibody-based methods, such as enzyme-linked immunosorbent assay (ELISA) and western blotting, have been used for decades. Unless arrays are adopted [91], their fitness to quantify proteins at a large scale level is poor. Conversely, MS-based quantification allows the identification of hundreds of proteins differentially expressed (DEPs) by both label [92] and label-free [93] approaches. ...

... Most existing techniques used for identifying specific substrates are enzyme-specific, labor-intensive, and often not straightforward. Such approaches include the use of genetic and pharmacologic perturbations 11 , substrate-trapping mutants 12 , affinity purification-mass spectrometry 13 , utilizing peptide 14 or protein arrays 15 , tagging the client proteins by substrate analogs using engineered enzymes 16 and peptide immunoprecipitation 17 or the employment of sophisticated computational tools 18 . Most of these techniques are specifically designed for a certain enzyme or enzyme class, which limits their applicability. ...

... Unicellular organisms and animal model systems, like Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, or Drosophila melanogaster have been the focus of attention in the first of these large-scale experiments . However the sequencing of the model plants Arabidopsis and rice have enabled large-scale approaches to also be conducted in plants (Arabidopsis Genome 2000; Bürkle et al. 2003; Fang et al. 2002; Goff et al. 2002; Kersten et al. 2002; Roberts 2002; Yu et al. 2002). The need for large-scale investigation of biological processes becomes especially evident when considering the high number of genes of unknown function and often inaccurate bioinformatic predictions. ...

... Proteins are immobilized on solid surface e.g. N-hydroxyl succinimide (NHS) derivatized slides or epoxy-and aldehyde-derivatized glass attachment through nitrocellulose (Petralia et al., 2017;Kramer et al., 2004), amines (Gerdtsson et al., 2016) or gel-coated slides (Charles et al., 2004) for attachment through absorption/adsorption, diffusion or nickel coated slides for affinity attachment of His-tagged proteins. The function of surface is not only the physical support but also should also demonstrate highest biding and maintain native conformation of proteins. ...

... The various strategies used include yeast-two hybrid screens, protein microarrays and in vitro kinase assays. Microarray-based testing of 1690 Arabidopsis proteins identified 48 in vitro substrates for MPK3 and 39 for MPK6, with an overlapping set of 26 candidates (Feilner et al., 2005). High-density protein microarrays used to determine the phosphorylation targets of 10 different MAPKs identified 570 potential MAPK targets out of 2158 candidates (Popescu et al., 2009). ...

... They range from in vivo approaches to in vitro approaches and may or may not allow precise localization of the phosphorylation sites on the proteins. These techniques will not be described here but are presented in numerous articles and reviews [45][46][47][48][49][50][51][52][53][54][55][56][57][58]. Once a precise phosphorylation site has been identified, however, two important questions remain: which particular protein kinase is responsible for the modification and what is the functional role of this modification. ...