A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background
A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background
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DOI：
10.1016/j.jim.2006.07.027
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发表时间：
2007-01-10
影响因子：
2.2
通讯作者：
Behrmann, Iris
中科院分区：
文献类型：
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作者：
Haan, Claude;Behrmann, Iris
我们比较了几种替代的ECL溶液，用于Western blot检测全细胞裂解物中的内源性蛋白质，使用廉价的、市售的试剂。从现有的基于对香豆酸（pCA）作为增强剂的方案出发，我们发现含有4-碘苯基硼酸（41PBA）的ECL溶液产生了强的特异性信号和低背景化学发光。我们优化了该41PBA- ecl溶液的lummol、41PBA和过氧化氢浓度。优化后的41PBA-ECL溶液（100 mM Tris/HCl pH 8.8, 1.25 mM鲁米诺，2 mM 41PBA， 5.3 mM过氧化氢）与最初的pCA-ECL方案和一些市售ECL溶液相比，信号强度大大提高。此外，与使用对香豆酸或4-碘酚作为增强剂的其他非商业ECL溶液相比，优化后的41PBA-ECL溶液产生的背景化学发光要低得多。41PBA-ECL溶液在储存时是稳定的，但从原液中新鲜制备时具有最低的背景。因此，我们提出了一种优化的协议，用于性能良好的廉价ECL解决方案，这是昂贵的商业ECL解决方案的替代方案，并且比测试的商业解决方案获得更好的信号和更低的背景。(c) 2006 Elsevier B.V.版权所有
We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (41PBA) generated strong specific signals and low background chemiluminescence. We optimised the lummol, 41PBA and hydrogenperoxide concentrations of this 41PBA-ECL solution. The optimised 41PBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 41PBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 41PBA-ECL solution also generates much lower background chemiluminescence than other noncommercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 41PBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested. (c) 2006 Elsevier B.V. All rights reserved.