The Third Xiangya Hospital, Central South University, Changsha, China.
Methods Mol Biol. 2023;2712:157-163. doi: 10.1007/978-1-0716-3433-2_14.
Ferroptosis is a regulated form of cell death caused by the excessive accumulation of iron-dependent lipid peroxidation. It has been implicated in various pathological processes and diseases, and its modulation involves multiple proteins associated with iron and lipid metabolism. To better understand these mechanisms and monitor the ferroptosis process, there is a need for reliable and high-throughput methods to evaluate variations in protein expression levels. In-Cell Western assays provide a simple and rapid assay method for detecting biomarkers and signaling proteins in whole cells using antibodies. This assay involves seeding cells in microtiter plates, followed by fixation/permeabilization and subsequent labeling with primary antibodies and infrared-conjugated secondary antibodies. In this chapter, we introduce the protocol for the In-Cell Western assay for detecting intracellular proteins during ferroptosis.
铁死亡是一种由铁依赖性脂质过氧化过度积累引起的受调控的细胞死亡形式。它与多种病理过程和疾病有关，其调节涉及与铁和脂质代谢相关的多种蛋白质。为了更好地理解这些机制并监测铁死亡过程，需要可靠的高通量方法来评估蛋白质表达水平的变化。In-Cell Western 检测法使用抗体在全细胞中检测生物标志物和信号蛋白的简单、快速的检测方法。该检测法包括在微量滴定板中接种细胞，然后进行固定/透化，接着用一抗和红外标记的二抗进行标记。在本章中，我们介绍了用于检测铁死亡过程中细胞内蛋白质的 In-Cell Western 检测法的实验方案。
