Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 432359
Title Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues.
Author(s) Smaczniak, C.; Li, N.; Boeren, J.A.; America, A.H.P.; Dongen, W.M.A.M. van; Goerdayal, S.S.; Vries, S.C. de; Angenent, G.C.; Kaufmann, K.
Source Nature protocols 7 (2012). - ISSN 1754-2189 - p. 2144 - 2158.
DOI https://doi.org/10.1038/nprot.2012.129
Department(s) Laboratory of Molecular Biology
Biochemistry
PRI BIOS Applied Genomics & Proteomics
PRI BIOS Plant Development Systems
EPS-1
Publication type Refereed Article in a scientific journal
Publication year 2012
Keyword(s) arabidopsis-thaliana - mass-spectrometry - quantitative proteomics - gene-expression - bac transgeneomics - quantification - strategy - reveals - cells
Abstract Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.
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