Protein depletion is a key approach to understanding the functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous/overexpressed TRIM21 to the antibody-bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of ~10-20 min. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies that are widely available; and that it can be applied to a wide range of cell types, including nondividing primary human cells, for which other loss-of-function assays are challenging. In this protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation, and for the generation of TRIM21-overexpressing cell lines for cases in which endogenous TRIM21 is limited. A typical Trim-Away experiment takes just a few hours.

中文翻译：

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Trim-Away 可快速快速降解内源性蛋白质。

蛋白质消耗是了解生物系统中蛋白质功能的关键方法。我们最近开发了 Trim-Away 方法，以便在没有事先修改的情况下快速降解内源性蛋白质。Trim-Away 基于泛素连接酶和 Fc 受体 TRIM21，可识别抗体结合蛋白并靶向它们以被蛋白酶体降解。在典型的 Trim-Away 实验中，蛋白质降解分三个步骤实现：首先，引入针对目标蛋白质的抗体；第二，将内源性或外源性/过表达的 TRIM21 募集到抗体结合的靶蛋白上；第三，蛋白酶体介导的靶蛋白、抗体和TRIM21复合物的降解。Trim-Away 对蛋白质的降解迅速而迅速，半衰期约为 10-20 分钟。与其他蛋白质降解方法相比，Trim-Away 的主要优势在于它可以应用于任何内源性蛋白质而无需事先修改；它使用广泛可用的常规抗体；并且它可以应用于广泛的细胞类型，包括不分裂的原代人类细胞，对于这些细胞，其他功能丧失检测具有挑战性。在本协议中，我们描述了通过显微注射和电穿孔在小鼠卵母细胞和培养细胞中制备和递送抗体的详细程序。此外，我们为抗体选择和验证提供建议，并为内源性 TRIM21 有限的情况下生成 TRIM21 过表达细胞系提供建议。一个典型的 Trim-Away 实验只需要几个小时。