一種新型蛋白質印跡檢測叫做雙印跡法，設計用于克服在免疫印跡實驗中第二抗體非特異性結合較高的問題。

這種方案使用Immobilon-P以及Millipore公司的PSQ轉移膜，將印跡蛋白轉移到Millipore的Immobilon膜。這種膜使用一抗標記，然后與第二張膜放在一起。兩張膜在酸性條件下進行第二次蛋白印跡檢測。

這種第二此印跡使得一抗分子從相應的抗原中解析出來，轉膜至第二張膜上，同時抗原與干擾蛋白質保留在第一張膜上。第二張膜從而可以使用第二抗體標記而沒有非特異性蛋白結合的干擾。

雙印跡法是由法國Chatanay-Malabry的國家反吸毒實驗室Francoise Lasne博士開發成功的。這種新方法應用范圍廣泛，適合各種研究目的，目前主要用于濃縮尿液中促紅細胞生成素的檢測。與傳統印跡方法相比較，雙印跡法最大程度地降低了生物素標記的二抗與尿液中蛋白的非特異性結合。

Millipore公司指出：Millipore的Immobilon-P以及PSQ是聚偏氟乙烯膜，具有統一控制的小孔結構以及較高的蛋白結合特性。與硝酸纖維素物質相比較，這些膜在雙印跡法所必須的酸性條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.