實驗概要
Peprotech provides a typical Western-transfer protocol (e.g. for the detection of protein X).
實驗原理
Western Transfer, also known as Western Blotting, is a rapid immunoblotting technique for identifying the presence of a particular protein in a complex mixture of proteins such as cell lysates or sera. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, and the ability of immunochemical reagents to interact specifically with a given protein antigen.
實驗步驟
1. The proteins in the tested solution are separated into distinct bands by SDS-PAGE,
2. The size-separated proteins are transferred from the polyacrylamide gel to a nitrocellulose membrane (or another suitable matrix).
3. The membrane containing the protein bands is serially incubated with:
1) a suitable blocking reagent to prevent non-specific protein binding,
2) a wash solution to rinse any unbound blocking reagent,
3) a probing antibody (anti-protein-X antibody) that forms a specific immune complex with Protein-X,.
4) additional wash solution to remove any unbound antibody,
5) an enzyme-linked antibody that binds specifically to the Fc region of the anti-Protein-X antibody,
6) additional wash solution to remove any unbound enzyme-linked antibody, and finally,
7) a substrate solution, which in the presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rendering the Protein X band visible (Figure III-1).
