The fastest Western in town: a contemporary twist on the classic Western blot analysis.
The fastest Western in town: a contemporary twist on the classic Western blot analysis.
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DOI：
10.3791/51149
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发表时间：
2014-02-05
期刊：
影响因子：
--
通讯作者：
McMahon M
中科院分区：
文献类型：
--
作者：
Silva JM;McMahon M
Western blot技术最初建立于20世纪70年代末，至今仍在积极应用。然而，这种传统的Western blotting方法有几个缺点，包括低质量分辨率，伪带，灵敏度降低，蛋白质完整性差。最近的进展大大改进了标准Western blot方案的许多方面，以产生更高的定性和定量数据。Bis-Tris凝胶系统是传统Laemmli系统的替代方案，可以产生更好的蛋白质分离和分辨率，保持蛋白质完整性，并将电泳时间缩短至35分钟。此外，iBlot干印迹系统在7分钟内显著提高了蛋白质转移到膜上的效率和速度，这与传统的蛋白质转移方法相比，传统的蛋白质转移方法通常效率较低，转移时间较长。结合这些高度创新的修改，与化学发光的标准Western印迹技术相比，使用红外荧光成像的蛋白质检测结果质量更高，更准确和一致。该技术利用在不同荧光通道中可见的双色近红外染料，可以同时检测同一膜上的两种不同抗原。此外，荧光成像的线性和宽动态范围允许对强和弱蛋白质带进行精确定量。因此，本协议描述了经典Western blotting方法的关键改进，其中这些改进显着提高了数据质量，同时大大缩短了实验的性能时间。
The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.
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DOI：
10.1073/pnas.76.7.3116
发表时间：
1979-01-01
影响因子：
11.1
作者：
RENART, J;REISER, J;STARK, GR
通讯作者：
STARK, GR
DOI：
10.1073/pnas.76.9.4350
发表时间：
1979-01-01
影响因子：
11.1
作者：
TOWBIN, H;STAEHELIN, T;GORDON, J
通讯作者：
GORDON, J
影响因子：
64.8
作者：
LAEMMLI, UK
通讯作者：
LAEMMLI, UK
影响因子：
2.2
作者：
DENHOLLANDER, N;BEFUS, D
通讯作者：
BEFUS, D
影响因子：
2.9
作者：
BURNETTE, WN
通讯作者：
BURNETTE, WN