Western blot analysis12/9/2023 NEVER inject a rabbit with complete adjuvant more than one time. Only use the complete adjuvant for the first inoculation. The complete adjuvant contains microbacteria (an immune stimulant) to increase the immune response. Crush it and make an emulsion with 1 mL Freund's Complete Adjuvant (which is an oily substance). This will allow you to see the protein band as a clear band against a milky white precipitate on the rest of the gel.Ĭarefully cut out the band and soak it in 1 mL PBS buffer. Since the area with the protein has a low concentration of SDS, the area with the protein will not show a precipitate. The KCl forms a precipitate with the SDS. This description assumes you have available purified protein. The reaction usually runs out in about an hour. Put x-ray film on your gel to detect a flash of light, which is given off by the enzyme. If everything worked properly, you will see bands wherever there is a protein-primary antibody-secondary antibody-enzyme complex, or, in other words, wherever your protein is.Ħ. To actually see your enzyme in action, you'll need to incubate it in a reaction mix that is specific for your enzyme. It's kind of like a molecular flare stuck on the antibodies so you can visualize what s going on.ĥ. The conjugated enzyme is there to allow you to visualize all of this. This means the secondary antibody will "stick" to the primary antibody, just like the primary antibody "stuck" to the protein. The secondary antibody should be an antibody against the primary antibody. This antibody should be an antibody-enzyme conjugate. Incubate the nitrocellulose membrane with a secondary 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.Ĥ. Click here to find out more about how to make a primary antibody. Incubate the nitrocellulose membrane with a primary antibody. You also need to be sure there are no air bubbles between the nitrocellulose and the gel or your proteins will not transfer.ģ. You may need to go through some trial-and-error to find the optimal pH. One thing to be aware of is that proteins bind better to nitrocellulose at a low pH. This gives you a nitrocellulose membrane that is imprinted with the same protein bands as the gel. 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. Place a nitrocellulose membrane on the gel and, using electrophoresis, drive the protein (polypeptide) bands onto the nitrocellulose membrane. Separate the proteins using SDS-polyacrylamide gel electrophoresis (also known as SDS-PAGE). Let's look at this technique in greater detail.ġ. See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques. Also, if a protein is degraded quickly, Western blotting won't detect it well you'll need to use (RIP). If you are interested in the rate of synthesis of a protein, Radio-Immune Precipitation (RIP) may be the best assay for you. Western blotting tells you how much protein has accumulated in cells. You will use this antibody as a probe to detect the protein of interest. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. It does not matter whether the protein has been synthesized in vivo or in vitro. 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.
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