Mount Desert Island Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salisbury Cove, ME 04609, USA.
Cell Rep Methods. 2022 Apr 25;2(4):100203. doi: 10.1016/j.crmeth.2022.100203.
The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in , they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact , we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.
通过蛋白质翻译来调节基因表达对于生长、发育和应激反应至关重要。虽然基于嘌呤霉素的技术已被用于定量 ，但其仅限于使用整个蠕虫的裂解物。为了在完整的 中实现核糖体活性的组织特异性定量，我们报告了在角质层缺陷突变体中应用 O-炔丙基嘌呤霉素，然后通过缀合叠氮荧光团进行检测，使用荧光共焦显微镜。我们将该技术应用于定量分析热休克、环己酰亚胺或翻译因子敲低时的翻译反应。此外，我们证明 O-炔丙基嘌呤霉素可用于定量组织之间或组织内（如生殖系）的翻译。预计该技术将在确定蛋白质翻译如何在不同组织中因应激、基因敲低或年龄而改变方面具有广泛的应用。
