16.2.1 Purification of ATeam Protein
ATeam protein was prepared according to the procedures by Imamura et al. (2009). First, Escherichia coli transformed by ATeam expression plasmid was cultured in LB medium overnight, then the collected bacteria were dissolved in buffer A [100 mM Na3PO4 (pH 8.0), 200 mM NaCl, and 10 mM imidazole]. After treatment by a sonicator, they were applied to a Ni-NTA column and eluted by buffer A with 200 mM imidazole. Furthermore, the concentrated ATeam fraction was purified by gel filtration and replaced in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. After adding glycerol to purified ATeam protein, it was frozen rapidly in liquid nitrogen and stored at −80 °C until use. Protein concentration was determined by the absorption at 515 nm of YFP.
16.2.2 Preparation of the Translucent Xenopus Oocytes
Xenopus oocytes and eggs contain pigment and yolk granules that interrupt the observation of fluorescence under microscopy. Therefore, the preparation of translucent oocytes or eggs is needed to observe the FRET signal of ATeam. In this report, we prepared the translucent oocytes using the methods by Iwao et al. (2005). Ovaries were dissected out from adult frogs (Xenopus laevis) and treated with 1 mg/ml collagenase. After washing, stage VI full-grown oocytes were stored in modified birth solution [MBS: 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, pH 7.6] with 0.01 % chloramphenicol. In advance, ATeam protein (23 or 46 nl, corresponding to 0.1 or 0.2 pmol, respectively) was injected into vegetal hemisphere of the oocytes. The oocytes were put onto 40 % Ficoll in MBS, then centrifuged at 3,000 g for 30 min (Fig. 16.1). After this treatment, lipids form a cap-like structure on the top of the oocytes; germinal vesicle may surround this lipid layer, and translucent cytoplasm occupies the remaining area in the animal hemisphere, while pigment granules set on the equator and yolk granules consists in whole vegetal hemisphere (Fig. 16.1). These translucent oocytes were put in MBS until observation.
16.2.3 Observation Under Microscopy and Image Analysis
The translucent oocytes were observed under fluorescence microscopy (Zeiss, Axioplan2) with the filters D480/30 for CFP and D535/40 for YFP (Photometrics, DualView2). The fluorescence images were taken by a digital camera (Hamamatsu, ORCA-Flash2.8) every 10 s. The fluorescence intensity was measured and then FRET signal of ATeam (YFP/CFP) was calculated using MetaMorph software (Molecular Devices). The results were shown as average intensity (Fig. 16.2), which is 1,000 fold of the FRET value. For the example in Fig. 16.2, 0.2 pmol ATeam protein was injected into an oocyte before treatment for translucent oocytes, then 9.2 nl 16.5 mM ATP solution was injected three times into an oocyte through a glass needle, which was inserted into the translucent cytoplasm beforehand.