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一种基于微孔板的非放射性蛋白质合成测定法：在蛋白质合成抑制剂对TRAIL致敏作用中的应用

A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

作者信息

Henrich Curtis J

机构信息

Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, 21702, United States of America.

Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 21702, United States of America.

出版信息

PLoS One. 2016 Oct 21;11(10):e0165192. doi: 10.1371/journal.pone.0165192. eCollection 2016.

DOI:10.1371/journal.pone.0165192
PMID:27768779
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5074477/
Abstract

Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.

摘要

基于嘌呤霉素掺入新合成蛋白质并随后使用抗嘌呤霉素抗体进行检测的非放射性测定方法此前已有报道且经过充分验证。为开发一种中高通量测定方法，本文描述了一种改进方法，即先对细胞进行嘌呤霉素标记，然后在原位同时检测嘌呤霉素标记的蛋白质和一种参考蛋白质。使用一对近红外标记的二抗进行检测（InCell western，ICW形式）可对384孔板中的蛋白质合成进行定量分析。优化后，将ICW结果与使用环己酰亚胺作为模型蛋白质合成抑制剂的蛋白质印迹分析结果进行比较，结果显示具有可比性。然后将该方法应用于几种蛋白质合成抑制剂，结果表明其作为蛋白质合成抑制剂的效力与其使TRAIL耐药性肾癌细胞对TRAIL诱导的凋亡敏感的能力之间具有良好的相关性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/74e83c999a38/pone.0165192.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/5e5836bb1e6b/pone.0165192.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/3adf429436fd/pone.0165192.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/74e83c999a38/pone.0165192.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/5e5836bb1e6b/pone.0165192.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/3adf429436fd/pone.0165192.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/33cf/5074477/74e83c999a38/pone.0165192.g003.jpg

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