Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol
Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol
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DOI：
10.1038/s41598-019-50385-9
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发表时间：
2019-09-26
影响因子：
4.6
通讯作者：
Biggar, Kyle K.
中科院分区：
文献类型：
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作者：
Chopra, Anand;Willmore, William G.;Biggar, Kyle K.
在聚丙烯酰胺凝胶中掺入2，2，2-三氯乙醇可使蛋白质在电泳后荧光可视化。紫外光照射，在这种三氯化化合物的存在下，导致色氨酸吲哚环的共价修饰，将荧光发射转移到可见光范围内。基于这一原理，我们使用2，2，2-三氯乙醇开发了基于修饰蛋白产生的荧光信号的微孔板形式的蛋白质定量分析。我们还证明了2，2，2-三氯乙醇标记的蛋白质在450 nm处的特定荧光发射，与310 nm的激发，从色氨酸和酪氨酸残基的修饰。优化后，与280 nm处的UV吸光度（A280）相比，该蛋白质定量测定显示出优越的上级灵敏度，并且能够在Bradford方法允许的线性范围外进行定量。该100 μ L测定在高达至少200 μ g的范围内显示出10.5 μ g的灵敏度。此外，我们通过开发20 μ L低容量测定法扩展了该方法的实用性，其灵敏度为8.7 μ g，最高可检测100 μ g，这使得SDS-PAGE后蛋白质可视化。总的来说，这些结果证明了基于2，2，2_三氯乙醇的蛋白质定量的实用性，并证明了基于2，2，2_三氯乙醇标记的预电泳的聚丙烯酰胺凝胶中的蛋白质可视化。
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-trichloroethanollabeled 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 mu L assay displayed a sensitivity of 10.5 mu g in a range up to at least 200 mu g. Furthermore, we extended the utility of this method through the development of a 20 mu L low-volume assay, with a sensitivity of 8.7 mu g tested up to 100 mu g, 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.