Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia.
Curr Protoc. 2023 Apr;3(4):e745. doi: 10.1002/cpz1.745.
Fluorescent proteins (FPs) have become an essential tool for biological research. Since the isolation and description of green FP, hundreds of FPs have been discovered and created with various characteristics. The excitation of these proteins ranges from ultraviolet (UV) up to near infrared (NIR). Using conventional cytometry, with each detector assigned to a fluorochrome, great care must be taken when selecting the optimal bandpass filters to minimalize the spectral overlap as the emission spectra of FPs are broad. Full-spectrum flow cytometers eliminate the need to change optical filters for analyzing FPs, which simplifies instrument setup. In experiments where more than one FP is used, single-color controls are required. These can be cells expressing each of the proteins separately. In the case of the confetti system, for instance, when four FPs are used, all these proteins will need to be expressed separately so that compensation or spectral unmixing can be performed, and this can be inconvenient and expensive. An appealing alternative is to produce FPs in Escherichia coli, purify them, and covalently couple them to carboxylate polystyrene microspheres. Such microspheres are ready to use and can be stored at 4°C for months or even years without any deterioration in fluorescence. The same procedure can be used to couple antibodies or other proteins to these particles. Here, we describe how to express and purify FPs, how to couple them to microspheres, and how to evaluate the fluorescent properties of the particles. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Escherichia coli expression and purification of recombinant mPlum Basic Protocol 2: Coupling fluorescent proteins to polystyrene microspheres Support Protocol 1: Comparing the cell-bound and bead-bound fluorescence signatures Support Protocol 2: Comparing spectral signatures via the similarity index, complexity matrix, and spillover spread matrix of fluorescent protein-coupled beads.
荧光蛋白（FPs）已成为生物学研究的重要工具。自绿色 FP 的分离和描述以来，已经发现并创建了数百种具有各种特性的 FP。这些蛋白质的激发范围从紫外线（UV）到近红外（NIR）。使用传统的细胞仪，每个检测器分配给一种荧光染料，在选择最佳带通滤波器以最小化 FP 发射光谱较宽的光谱重叠时必须非常小心。全谱流式细胞仪无需更换光学滤波器即可分析 FP，从而简化了仪器设置。在使用多个 FP 的实验中，需要使用单颜色对照。这些可以是分别表达每种蛋白质的细胞。例如，在 confetti 系统的情况下，当使用四个 FP 时，所有这些蛋白质都需要单独表达，以便可以进行补偿或光谱解混，这可能既不方便又昂贵。一个有吸引力的替代方案是在大肠杆菌中表达 FP，对其进行纯化，并将其共价偶联到羧基化聚苯乙烯微球上。这些微球可即用于实验，可以在 4°C 下储存数月甚至数年，而荧光不会恶化。同样的程序可以用于将抗体或其他蛋白质偶联到这些颗粒上。在这里，我们描述了如何表达和纯化 FP，如何将它们偶联到微球上，以及如何评估颗粒的荧光特性。 © 2023 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：大肠杆菌表达和纯化重组 mPlum 基本方案 2：将荧光蛋白偶联到聚苯乙烯微球上 支持方案 1：比较细胞结合和珠结合荧光特征 支持方案 2：通过荧光蛋白偶联珠的相似指数、复杂度矩阵和溢出扩散矩阵比较光谱特征。
