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An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes

(2015) NATURE PROTOCOLS. 10(1). p.169-187
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Bioinformatics: from nucleotids to networks (N2N)
Abstract
Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (similar to 7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; similar to 5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.
Keywords
FLORAL DIP, TRANSCRIPTION, SACCHAROMYCES-CEREVISIAE, LEAF DEVELOPMENT, DIVISION, PROTEOMICS, THALIANA, AGROBACTERIUM-MEDIATED TRANSFORMATION, SUSPENSION-CULTURES, ANAPHASE-PROMOTING COMPLEX/CYCLOSOME

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Chicago
Van Leene, Jelle, Dominique Eeckhout, Bernard Cannoot, Nancy De Winne, Geert Persiau, Eveline Van De Slijke, Leen Vercruysse, et al. 2015. “An Improved Toolbox to Unravel the Plant Cellular Machinery by Tandem Affinity Purification of Arabidopsis Protein Complexes.” Nature Protocols 10 (1): 169–187.
APA
Van Leene, J., Eeckhout, D., Cannoot, B., De Winne, N., Persiau, G., Van De Slijke, E., Vercruysse, L., et al. (2015). An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. NATURE PROTOCOLS, 10(1), 169–187.
Vancouver
1.
Van Leene J, Eeckhout D, Cannoot B, De Winne N, Persiau G, Van De Slijke E, et al. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. NATURE PROTOCOLS. 2015;10(1):169–87.
MLA
Van Leene, Jelle, Dominique Eeckhout, Bernard Cannoot, et al. “An Improved Toolbox to Unravel the Plant Cellular Machinery by Tandem Affinity Purification of Arabidopsis Protein Complexes.” NATURE PROTOCOLS 10.1 (2015): 169–187. Print.
@article{5835945,
  abstract     = {Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (similar to 7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; similar to 5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.},
  author       = {Van Leene, Jelle and Eeckhout, Dominique and Cannoot, Bernard and De Winne, Nancy and Persiau, Geert and Van De Slijke, Eveline and Vercruysse, Leen and Dedecker, Maarten and Verkest, Aurine and Vandepoele, Klaas and Martens, Lennart and Witters, Erwin and Gevaert, Kris and De Jaeger, Geert},
  issn         = {1754-2189},
  journal      = {NATURE PROTOCOLS},
  keyword      = {FLORAL DIP,TRANSCRIPTION,SACCHAROMYCES-CEREVISIAE,LEAF DEVELOPMENT,DIVISION,PROTEOMICS,THALIANA,AGROBACTERIUM-MEDIATED TRANSFORMATION,SUSPENSION-CULTURES,ANAPHASE-PROMOTING COMPLEX/CYCLOSOME},
  language     = {eng},
  number       = {1},
  pages        = {169--187},
  title        = {An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes},
  url          = {http://dx.doi.org/10.1038/nprot.2014.199},
  volume       = {10},
  year         = {2015},
}

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