MPI für molekulare Genetik / Department of Vertebrate Genomics |
|Generation of Arabidopsis protein chips for antibody and serum screening|
|Authors:||Kersten, Birgit; Feilner, Tanja; Kramer, A.; Wehrmeyer, Silke; Possling, Alexandra; Witt, Iris; Zanor, María-Inés; Stracke, Ralf; Lueking, Angelika; Kreutzberger, Juergen; Lehrach, Hans; Cahill, Dolores J.|
|Date of Publication (YYYY-MM-DD):||2003-07|
|Title of Journal:||Plant Molecular Biology|
|Journal Abbrev.:||Plant Mol.Biol.|
|Issue / Number:||5|
|Copyright:||Copyright © 2003 Kluwer Academic Publishers,
All rights reserved
|Review Status:||not specified|
|Abstract / Description:||Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far.
The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2–3.6 fmol per spot on FAST slides or 0.1–1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.
|Free Keywords:||Arabidopsis thaliana, Escherichia coli, high-throughput, protein micro-array, proteomics, transcription factor|
|Comment of the Author/Creator:||Date: 2003, JUL|
|External Publication Status:||published|
|Version Comment:||Automatic journal name synchronization|
|Communicated by:||Hans Lehrach|
|Affiliations:||MPI für molekulare Genetik|
|External Affiliations:||Max Planck Inst Mol Genet, D-14195 Berlin, Germany.; Max Planck Inst Mol Plant Physiol, D-14476 Golm, Germany.; Max Planck Inst Plant Breeding Res, D-50829 Cologne, Germany.|
|Identifiers:||ISI:000185028800007 [ID No:1] |
ISSN:0167-4412 [ID No:2]
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