Western blotting

Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. You will use this antibody as a probe to detect the protein of interest.

Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-Immune Precipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won't detect it well; you'll need to use (RIP). See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques.

Let's look at this technique in greater detail.

1. Separate the proteins using SDS-polyacrylamide gel electrophoresis (also known as SDS-PAGE). This separates the proteins by size.

2. Place a nitrocellulose membrane on the gel and, using electrophoresis, drive the protein (polypeptide) bands onto the nitrocellulose membrane. You want the negative charge to be on the side of the gel and the positive charge to be on the side of the nitrocellulose membrane to drive the negatively charged proteins over to the positively charged nitrocellulose membrane. This gives you a nitrocellulose membrane that is imprinted with the same protein bands as the gel.

One thing to be aware of is that proteins bind better to nitrocellulose at a low pH. You may need to go through some trial-and-error to find the optimal pH. You also need to be sure there are no air bubbles between the nitrocellulose and the gel or your proteins will not transfer.

3. Incubate the nitrocellulose membrane with a primary antibody. Click here to find out more about how to make a primary antibody. The primary antibody, which is the specific antibody mentioned above, sticks to your protein and forms an antibody-protein complex with the protein of interest.