Department of Biology, Carleton University, 1125 Colonel By Dr, Ottawa, ON, K1S 5B6, Canada.
Institute of Biochemistry, Carleton University, 1125 Colonel By Dr, Ottawa, ON, K1S 5B6, Canada.
Sci Rep. 2019 Sep 26;9(1):13923. doi: 10.1038/s41598-019-50385-9.
The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.
2,2,2-三氯乙醇在聚丙烯酰胺凝胶中的掺入允许在电泳后对蛋白质进行荧光可视化。在存在这种三氯化合物的情况下，紫外线照射会导致色氨酸吲哚环的共价修饰，从而将荧光发射转移到可见范围。基于这一原理，我们使用 2,2,2-三氯乙醇开发了一种微孔板格式的蛋白质定量测定法，该方法基于修饰蛋白产生的荧光信号。我们还证明了 2,2,2-三氯乙醇标记蛋白在 450nm 处具有特定的荧光发射，激发波长为 310nm，这是由于色氨酸和酪氨酸残基的修饰。经过优化，与 280nm 处的紫外吸收（A280）相比，这种蛋白质定量测定法显示出更高的灵敏度，并且能够在 Bradford 方法允许的线性范围之外进行定量。这种 100μL 测定法在 10.5μg 至至少 200μg 的范围内具有灵敏度。此外，我们通过开发 20μL 低体积测定法扩展了该方法的实用性，其灵敏度为 8.7μg，测试范围高达 100μg，这使得 SDS-PAGE 后能够可视化蛋白质。总之，这些结果证明了基于 2,2,2-三氯乙醇的蛋白质定量测定的实用性，并证明了基于 2,2,2-三氯乙醇标记预电泳的聚丙烯酰胺凝胶中的蛋白质可视化。
