Leibniz Institute on Aging, Fritz Lipmann Institute, 07745, Jena, Germany.
Anal Biochem. 2020 Mar 15;593:113598. doi: 10.1016/j.ab.2020.113598. Epub 2020 Feb 1.
Blocking conditions are critical for reducing background and non-specific signals in Western blot (WB). For fluorescent WB, however, blockers may bring in additional autofluorescence, and optimal blocking conditions have been less well established. Here, the autofluorescence of Tween 20 is clarified, which is negligible when imaged wet, but could be significant when Tween 20 is dried on blots. In most situations, blocking with Tween 20 reduces background and/or improves specific signals. Systematical investigation of blocking conditions for fluorescent WB reveals that the autofluorescent background is determined by a combination of multiple factors including the blocker, the excitation/emission configuration, the membrane, and the HO (wet or dry), varying case by case. The commonly used protein or polymer blockers (milk, bovine serum albumin, fish gelatin, and polyvinylpyrrolidone) each have unique advantages and disadvantages concerning the autofluorescence, the blocking efficiency, and the cost. 0.005%-0.02% sodium dodecyl sulphate can be included to reduce non-specific bands and background caused by non-specific binding of antibodies, however, at the cost of also impairing specific signals. For poorly-performing antibodies, a pre-test might be necessary to identify a suitable blocker. This work provides a reference for cost-effective blocking conditions for fluorescent WB.
阻断条件对于减少 Western blot（WB）中的背景和非特异性信号至关重要。然而，对于荧光 WB，阻断剂可能会带来额外的自发荧光，并且优化的阻断条件尚未得到很好的建立。在这里，澄清了吐温 20 的自发荧光，在湿状态下成像时可以忽略不计，但在吐温 20 干燥在印迹上时可能会很显著。在大多数情况下，用吐温 20 阻断可以减少背景和/或改善特异性信号。对荧光 WB 阻断条件的系统研究表明，自发荧光背景由多种因素共同决定，包括阻断剂、激发/发射配置、膜和 HO（湿或干），具体情况具体分析。常用的蛋白质或聚合物阻断剂（牛奶、牛血清白蛋白、鱼明胶和聚乙烯吡咯烷酮）在自发荧光、阻断效率和成本方面各有优缺点。0.005%-0.02%的十二烷基硫酸钠可以包含在内，以减少由于抗体的非特异性结合引起的非特异性条带和背景，但这也会损害特异性信号。对于性能不佳的抗体，可能需要进行预测试以确定合适的阻断剂。这项工作为荧光 WB 的经济有效的阻断条件提供了参考。
