Preparing the Plant Material

It has been previously mentioned that fusion proteins should be isolated preferentially from transgenic plants cultivated under specific growth conditions (temperature, luminosity, humidity, media composition, etc.) in which fully functionality of tagged proteins has been confirmed. In this scenario, we will be able to assume that regulation of the biochemical activity, map of protein interactions, and subcellular localization of a particular fusion resembles that of the endogenous protein. Once growth conditions for a specific transgenic line have been chosen, accumulation of the recombinant protein should be assessed in a time course experiment. A compromise ought to be reached between peak accumulation of tagged proteins and amount of fresh tissue. Thus, it is possible that a particular tagged protein accumulates at its highest in 5-day-old seedlings and only a half of these levels remain after two weeks. However, 2-week-old seedlings may provide about 10 times more fresh material than 5-day-old plants, thus containing a higher amount of total tagged protein.

Regarding the amount of fresh plant material necessary for successful TAPa purification, it generally depends on the level of expression of each tagged protein. Our experience has shown us that purification of silver staining-detectable levels of a multimeric protein complex can be accomplished by using just 1.5 g of Arabidopsis fresh tissue [7, 8]. However, this is an optimal situation and, in general, more than 10 g plant material is necessary to obtain MS detectable levels of tagged protein.

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