双分⼦荧光互补BiFC对照设置详细（revised） 单水合肼内部对照也是必要的，可以标准化转染效率和蛋⽩在不同细胞中表达的差异。这个对照的完成是由，编码含⽬的蛋⽩的融合蛋⽩和完整的⾮⽚段蛋⽩（这个完整的蛋⽩⾥的荧光报告蛋⽩在不同的波长下发荧光）的质粒共转染细胞。在可视化过程 链式运输机中，one（其中⼀个蛋⽩，融合或完整）来确定BiFC复合体的荧光强度，和在消除背景信号后内部对照变成⼀个⽐值。这个⽐值代表了，BiFC效率，并可以与其他⽐值对⽐以确定其他形式的不同复合物的相对效率。 注意：细胞和质粒都是复数形式，意思是⽤多个相同质粒转染多个相同细胞，这样可以标准化转染效率！
在其他⽂献上看到⼀个图，就不翻译了这个可能好懂很多。见下页：
塑料标签Figure 4. Two key elements of a BiFC experiment: fusionorientation(融合⽅向) and controls. (A) Each o
f the two YFP halves (YFP N and YFP C) can be fused at either its N- or C-terminus, with the protein of interest. Likewise, the protein of interest can also be fused to the YFP half via its N- or C-terminus. This creates four possible YFP-protein combinations for each protein of interest and eight combinations that should be tested for each interaction pair; (B) In addition to the protein-protein interaction to be tested, (i) appropriate negative controls should be incorporated in a BiFC experiment; These negative controls include substitution for one of the protein of interest by (ii) a mutated protein (mP1); (iii) a related protein (PX) that does not interact; and (iv) an unrelated protein (PY) with the same subcellular localization. The stars indicate YFP fluorescence due to self-assembly (one star) and expression due to a bona fide（⼀概⽽论）interaction between the test proteins (three stars).
划线仪
长途运输鱼苗时显示器自动开关机上述Cited from A Cautionary Note on the Use of Split-YFP/BiFC in Plant Protein-Protein Interaction Studies
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