Teng I-Ting, Bu Xiangning, Chung Inhee
Department of Anatomy and Cell Biology, George Washington University, School of Medicine and Health Sciences, Washington, District of Columbia, USA.
Bio Protoc. 2019 Sep 20;9(18):e3375. doi: 10.21769/BioProtoc.3375.
Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have become valuable tools to discern how endogenous proteins control cell biology. Yet, some technical challenges remain; in SMT, these revolve around the characteristics of the labeling reagent. A good reagent should have neutrality (in terms of not affecting the target protein's functions), tagging specificity, and a bright fluorescence signal. In addition, a long shelf-life is desirable due to the time and monetary costs associated with reagent preparation. Semiconductor-based quantum dots (Qdots) or Janelia Fluor (JF) dyes are bright and photostable, and are thus excellent candidates for SMT tagging. Neutral, high-affinity antibodies can selectively bind to target proteins. However, the bivalency of antibodies can cause simultaneous binding to two proteins, and this bridging effect can alter protein functions and behaviors. Bivalency can be avoided using monovalent Fab fragments generated by enzymatic digestion of neutral antibodies. However, conjugation of a Fab with a dye using the chemical cross-linking agent SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) requires reduction of the interchain disulfide bond within the Fab fragment, which can decrease the structural stability of the Fab and weaken its antigen-binding capability. To overcome this problem, we perform limited reduction of F(ab')2 to generate Fab' fragments using a weak reducer, cysteamine, which yields free sulfhydryl groups in the hinge region, while the interchain disulfide bond in Fab' is intact. Here, we describe a method that generates Fab' with high yield from two isoforms of IgG and conjugates the Fab' fragments with Qdots. This conjugation scheme can be applied easily to other types of dyes with similar chemical characteristics.
随着先进光学成像技术的出现，我们对细胞表面蛋白的调控和功能的理解有了迅速进展。特别是，使用与抗体偶联的明亮荧光团的单分子追踪（SMT）以及诸如全内反射荧光显微镜等宽场显微镜方法，已成为识别内源性蛋白如何控制细胞生物学的有价值工具。然而，一些技术挑战仍然存在；在SMT中，这些挑战围绕着标记试剂的特性。一种好的试剂应具有中性（即不影响靶蛋白的功能）、标记特异性和明亮的荧光信号。此外，由于试剂制备相关的时间和金钱成本，希望其具有较长的保质期。基于半导体的量子点（Qdots）或Janelia Fluor（JF）染料明亮且光稳定，因此是SMT标记的极佳候选物。中性、高亲和力抗体可选择性结合靶蛋白。然而，抗体的二价性可导致同时结合两种蛋白，这种桥接效应可改变蛋白的功能和行为。使用通过中性抗体酶切产生的单价Fab片段可避免二价性。然而，使用化学交联剂SMCC（琥珀酰亚胺基4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯）将Fab与染料偶联需要还原Fab片段内的链间二硫键，这会降低Fab的结构稳定性并削弱其抗原结合能力。为克服这一问题，我们使用弱还原剂半胱胺对F(ab')2进行有限还原以生成Fab'片段，半胱胺在铰链区产生游离巯基，而Fab'中的链间二硫键保持完整。在此，我们描述了一种从两种IgG同工型高产率生成Fab'并将Fab'片段与Qdots偶联的方法。这种偶联方案可轻松应用于具有类似化学特性的其他类型染料。
