Test001/Notebook/Fragaceatoxin C

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μL==Experimental procedures==

Protein purification

FraC was isolated from specimens of Actinia fragacea collected from the shoreline of the northern rocky coast of Spain, facing the Cantabrian sea and the Bay of Biscay (figure 4.4a). The purification protocol is described elsewhere (Bellomio et al., 2009). It is largely based on the isolation of recombinant equinatoxin II (Anderluh et al, 1996), which avoids the acetone-precipitation step described for the purification of natural equinatoxin II (Macek and Lebez, 1988). This method maintains the toxin in its native conformation and minimizes the protein loss that inevitably takes place after acetone precipitation.

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Protein crystallization

Initial crystallization screenings (192 conditions) were performed using the sitting-drop method in 96-well CrystalQuick plates, dispensing 200 nl drops using a Mosquito robot (TTP LabTech). Preliminary results performed at room temperature with a 1:1 mixture of protein solution (6 mg/ml) and 1.28 M sodium malonate, 0.11% LDAO pH 7.0 (condition G5 of the High Probability Salt Screen from Axygen Biosciences) gave very thin plate-shaped crystals (figure 4.5).