通过免疫印迹法检测单个同工tRNA上mG修饰的实验方案。

Protocol to detect mG modification on single tRNA iso-decoders by immuno-northern blotting.

作者信息

Hwang Sseu-Pei, Barondeau Katherine, Denicourt Catherine

机构信息

Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center, Houston, TX 77030, USA; The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX 77030, USA.

Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center, Houston, TX 77030, USA.

出版信息

STAR Protoc. 2025 Jul 1;6(3):103924. doi: 10.1016/j.xpro.2025.103924.

DOI:10.1016/j.xpro.2025.103924

PMID:40601427

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12268536/

Abstract

tRNA modifications are highly dynamic and play crucial roles in regulating gene expression. Here, we present an assay for analyzing N, N-dimethylguanosine (mG) modifications on tRNAs using an antibody-based detection approach. We describe steps for isolating tRNAs from human cells via ion-exchange chromatography, capturing specific tRNA iso-decoders on streptavidin magnetic beads, and detecting modifications through immuno-northern blotting. This approach enables researchers to investigate other tRNA modifications using specific antibodies or downstream approaches such as mass spectrometry. For complete details on the use and execution of this protocol, please refer to Hwang et al..

摘要

转运RNA（tRNA）修饰具有高度动态性，在调节基因表达中发挥关键作用。在此，我们展示了一种使用基于抗体的检测方法分析tRNA上N，N-二甲基鸟苷（mG）修饰的检测方法。我们描述了通过离子交换色谱从人细胞中分离tRNA、在链霉亲和素磁珠上捕获特定tRNA同工解码器以及通过免疫印迹检测修饰的步骤。这种方法使研究人员能够使用特异性抗体或质谱等下游方法研究其他tRNA修饰。有关本方案使用和执行的完整详细信息，请参阅Hwang等人的文献。