利用CG-SLENP对新合成蛋白和现有蛋白进行双重标记

IF 2.2
Bingbing X. Li, Xiangshu Xiao
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引用次数: 0

摘要

本文描述了一种鲁棒的新合成蛋白和现有蛋白双重标记方法。由于难以特异性地分离它们,因此从大量蛋白质组中评估单个新合成蛋白质的特性具有挑战性。以前的方法,如非天然氨基酸标记,同位素标记的氨基酸标记和嘌呤霉素标记是有效的系综测量。然而,这些现有的方法不支持活细胞跟踪或对特定靶蛋白的动态研究。我们设计了一种基于化学遗传学的方法来选择性标记现有和新合成的蛋白质(CG-SLENP)。以HaloTag标记的核层蛋白A (LA) (HaloTag-LA)为例蛋白和各种Halo配体,我们描述了CG-SLENP用于标记现有蛋白和新合成的蛋白。这种方法可以一次标记一个蛋白质,也可以在同一个活细胞中标记两个蛋白质。这种方法在研究任何感兴趣的特定蛋白质方面具有更广泛的应用潜力。©2025 Wiley Periodicals llc .基本协议1:使用可点击的Halo配体标记CG-SLENP基本协议2:使用荧光Halo配体标记CG-SLENP
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using CG-SLENP

Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using CG-SLENP

Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using CG-SLENP

Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using CG-SLENP

This article describes a robust method for dual labeling of newly synthesized proteins and existing proteins. Assessing the properties of individual newly synthesized proteins from the bulk proteome is challenging due to difficulty in specifically isolating them. Previous methods such as non-natural amino acid labeling, isotope-labeled amino acid labeling, and puromycin labeling are effective for ensemble measurements. However, these existing methods do not support live-cell tracking or dynamic studies for a specific target protein. We designed a chemical genetics–based method for selective labeling of existing and newly synthesized proteins (CG-SLENP). Using nuclear lamin A (LA) tagged with a HaloTag (HaloTag-LA) as an exemplar protein and various Halo ligands, we describe CG-SLENP for labeling existing proteins and newly synthesized proteins. This approach can label these proteins either one at a time or dually in the same live cell. This method holds great potential for broader applications to study any given protein of interest.©2025 Wiley Periodicals LLC.

Basic Protocol 1: CG-SLENP labeling using clickable Halo ligands

Basic Protocol 2: CG-SLENP labeling using fluorescent Halo ligands

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