Regardless of whether the protein in question has been synthesized in vivo (naturally) or in vitro (in the laboratory), Du Pont Western Blot techniques are able to detect one protein in a mixture of any number of proteins. They provide information about the size of the protein in question. The method is, however, dependent on the provision of a high-quality antibody directed against the desired protein. A small portion of the protein must be able to be created from a cloned DNA fragment, and this antibody is used to detect the protein of interest.
First, the proteins must be separated by size through use of the gel electrophoresis technique. Next, a nitrocellulose membrane must be placed on the gel, and the electrophoresis should be employed to drive the protein bands onto the nitrocellulose membrane. Following this, the nitrocellulose membrane is incubated using a primary antibody, which sticks to the protein, then with a secondary antibody. This secondary antibody is used as an antibody-enzyme conjugate, and is intended to stick to the primary antibody. The conjugated enzyme functions to help visualize what is occurring.
In the Du Pont Western Blot technique, the cloned enzyme can be seen in action by using the Radioactive Detection method. First, the cloned enzyme should be incubated in a reaction mix that is specific to the enzyme. If the procedure is undertaken properly, bands should become evident in the gel wherever the protein is in evidence. An X-ray film can be placed on the gel to detect flash of light that is given off by the enzyme.
Colorimetric Detection depends on the incubation of the Western Blot with a substrate designed to react with the reporter enzyme, bound to the secondary antibody. This results in the conversion of the soluble dye into an insoluble form exhibiting a different color that stains the nitrocellulose membrane.
In the Fluorescent Detection method, a labeled probe is excited by light, and the emission of the excitation is subsequently detected by a photosensor equipped with appropriate emission filters. This captures a digital image of the Western Blot and enables further data analysis. It is considered to be one of the most sensitive Western Blot detection methods currently available.
Before adding the primary antibody, the nitrocellulose gel should be incubated using a blot. Bovine serum albumin (BSA) is often used, but, perhaps surprisingly, Carnation Nonfat Dry Milk makes one of the best blotting agents.