免疫荧光通过荧光团标记的抗体定位蛋白质。然而,一些蛋白质由于抗体可及性问题或由于它们天然丰度低或抗原密度被成像方法降低而逃避检测。这里,我们表明,目标蛋白与生物素连接酶TurboID的融合以及随后通过荧光链霉亲和素检测生物素化提供了这些限制的“全部”解决方案。对于所有测试的蛋白质,链霉亲和素信号明显强于抗体信号,显着提高了扩展显微镜和相关的光学和电子显微镜的灵敏度。重要的是,相分离区域内的蛋白质,例如核孔的中心通道,核仁,或者RNA颗粒,很容易用链霉亲和素检测到,而大多数抗体都失败了。当TurboID与HA表位标签串联使用时,与链霉亲和素和抗HA共同探测可以绘制抗体可及性图,我们为锥虫核孔创建了这样的图。最后,我们表明,链霉亲和素成像解决了动态,暂时,和空间上不同的子复合体,在特定情况下,揭示了动态蛋白质相互作用的历史。总之,链霉亲和素成像对于检测低丰度或不可接近的蛋白质具有主要优势,提供有关蛋白质相互作用和生物物理环境的信息。
Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent
streptavidin offers an \'all in one\' solution to these restrictions. For all proteins tested, the
streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with
streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that
streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion,
streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.