新型蛋白质双印迹检测方法开发成功

一种新型蛋白质印迹检测叫做双印迹法，设计用于克服在免疫印迹实验中第二抗体非特异性结合较高的问题。

这种方案使用Immobilon-P以及Millipore公司的PSQ转移膜，将印迹蛋白转移到Millipore的Immobilon膜。这种膜使用一抗标记，然后与第二张膜放在一起。两张膜在酸性条件下进行第二次蛋白印迹检测。

这种第二此印迹使得一抗分子从相应的抗原中解析出来，转膜至第二张膜上，同时抗原与干扰蛋白质保留在第一张膜上。第二张膜从而可以使用第二抗体标记而没有非特异性蛋白结合的干扰。

双印迹法是由法国Chatanay-Malabry的国家反吸毒实验室Francoise Lasne博士开发成功的。这种新方法应用范围广泛，适合各种研究目的，目前主要用于浓缩尿液中促红细胞生成素的检测。与传统印迹方法相比较，双印迹法最大程度地降低了生物素标记的二抗与尿液中蛋白的非特异性结合。

Millipore公司指出：Millipore的Immobilon-P以及PSQ是聚偏氟乙烯膜，具有统一控制的小孔结构以及较高的蛋白结合特性。与硝酸纤维素物质相比较，这些膜在双印迹法所必须的酸性条件下具有可靠的特性。

New Western Blotting Method

A new western blotting technique called Double Blotting is designed to overcome the high nonspecific binding of secondary antibodies during immunoblotting.

The procedure, which uses the Immobilon-P and PSQ transfer membranes of Millipore (Billerica, MA, USA), involves blotting proteins onto a sheet of Millipore’s Immobilon membrane. The membrane is probed with the primary antibody and then assembled with a second membrane sheet. Both membrane sheets undergo a second blotting under acidic conditions.

This second blot allows the primary antibody molecules to desorb from their corresponding antigen and transfer onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first membrane. The second membrane can then be probed by secondary antibodies without risk of nonspecific binding.

The Double Blotting method was developed by Dr. Francoise Lasne of the National Anti-Doping Laboratory in Chatanay-Malabry (France). The new method is suitable for a wide range of research applications, and is currently used for the detection of erythropoietin (EPO) in concentrated urine. Compared to traditional blotting, Double Blotting minimizes strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins.

Millipore’s Immobilon-P and PSQ are polyvinylidene fluoride (PVDF) membranes, with a uniformly controlled pore structure and a high protein-binding capacity. In contrast to nitrocellulose materials, these membranes demonstrate reliable performance under the acidic conditions needed for Double Blotting, states Millipore.