WESTERN BLOTTING OF PROTEINS
Often researchers will use Western blotting to identify proteins. Western blots rely on having an antibody to the protein. Antibodies are extremely specific and will bind only to one target protein.
The first step is to separate the proteins by size by either standard SDS-PAGE or 2D-PAGE. The proteins are then transferred from the gel to a type of paper membrane made of nitrocellulose. Other types of membranes are made from nylon and are stronger. Either way, the membrane must have a positive charge so that the negatively charged proteins will stick to its surface. The proteins are moved from the gel to the membrane with an electric current as shown in Fig. 9.4.
After the proteins are attached to the nitrocellulose membrane, the primary antibody is used to find the location of the target protein. However, many areas of the membrane will not have any protein bound, because the corresponding area of the protein gel was empty. These blank areas are positively charged and can bind nonspecifically to the antibody. Therefore, these sites must be blocked. Often, the membranes are soaked in reconstituted nonfat dry milk. The milk proteins mask the unused sites on the membrane and will not bind to the antibody. Next, the antibody is added to the membrane in a solution. The antibody will bind only to its target protein, and nowhere else on the membrane, because it recognizes only one specific epitope of its target protein.
The next step is to visualize the location of the primary antibody, thus revealing the location of the target protein (Fig. 9.5). To achieve this, a secondary antibody is added. This antibody recognizes the stem of the primary antibody without affecting its binding to the protein. The secondary antibody has a tag or label on its stem that is easily detected. Often the tag is the enzyme alkaline phosphatase, which removes phosphate from various substrates. If the antibody complex is incubated with a chromogenic substrate such as X-Phos, the alkaline phosphatase removes the phosphate from the X-Phos. The remaining indolyl group reacts with oxygen to form a blue precipitate in the location of the target protein. If a chemiluminescent substrate is used, the nitrocellulose membrane is placed next to a piece of photographic film. The light pulses turn the film black where the secondary antibody/primary antibody/target protein complex is located on the membrane. Western blots are used extensively both to prove that a protein is being expressed and to estimate its level, because the intensity of the spot directly correlates with the amount of protein.