MacTaggart Brittany, Kashina Anna S
Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2023;2620:81-85. doi: 10.1007/978-1-0716-2942-0_10.
In this chapter, we present a simplified version of the method described in Chapter 9 of this book, adapted for fast and convenient evaluation of intracellular arginylation activity in live cells. As in the previous chapter, this method utilizes a GFP-tagged N-terminal β-actin peptide transfected into cells as a reporter construct. Arginylation activity can then be evaluated by harvesting the reporter-expressing cells and analyzing them directly by Western blot using an arginylated β-actin antibody and a GFP antibody as an internal reference. While absolute arginylation activity cannot be measured in this assay, different types of reporter-expressing cells can be directly compared, and the effect of genetic background or treatment can be evaluated. For its simplicity and broad biological application, we felt this method merited presentation here as a separate protocol.
在本章中，我们展示了本书第9章所述方法的简化版本，该版本适用于快速便捷地评估活细胞内的精氨酰化活性。与上一章一样，此方法利用转染到细胞中的绿色荧光蛋白（GFP）标记的N端β-肌动蛋白肽作为报告构建体。然后，通过收获表达报告基因的细胞，并使用精氨酰化β-肌动蛋白抗体和GFP抗体作为内部对照，直接通过蛋白质印迹法对其进行分析，从而评估精氨酰化活性。虽然在此测定中无法测量绝对精氨酰化活性，但可以直接比较不同类型的表达报告基因的细胞，并评估遗传背景或处理的效果。鉴于其简单性和广泛的生物学应用，我们认为此方法值得作为一个单独的方案在此处介绍。
