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

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants. The protein of interest is fused to a double affinity tag, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of an optimized TAP-tag combined with ultrasensitive MS enabled TAP on Arabidopsis cell cultures with relatively low biomass input. Moreover, we were able to extend the isolation of low abundant protein complexes toward Arabidopsis seedlings, opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (~6 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (~5 d), either from Arabidopsis seedlings or cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of non-specific proteins, based on 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.

Van Leene, J., Eeckhout, D., Cannoot, B., De Winne, N., Persiau, G., Van De Slijke, E., Vercruysse, L., Dedecker, M., Verkest, A., Vandepoele, K., Martens, L., Witters, E., Gevaert, O., De Jaeger, G. (2015) An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. Nature Protocols 10(1):169-87.

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