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Re: Western blot
hi i want to sare a article on Western blot:
Western blotting is a technique used to identify and locate proteins based on their ability to bind to specific antibodies.
Western blot analysis can detect your protein of interest from a mixture of a great number of proteins. Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue).
Summary: Western Blot Gives You Information on the:
# Size of your Protein
# Expression Amount of your Protein
Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro. Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein. Antibodies are now easily obtainable from commercial sources, and you can purchase one for your protein of interest. If your protein is a novel protein, you must produce an antibody yourself or get a company to do it for you. In this case you will need at least a small amount of your protein either purified from cell extracts or made as a recombinant (ie in vitro or in a recombinant protein expression system). Antibodies specific to your protein are vital to western blotting as they are able to bind specifically to your protein of interest instead of the thousands of proteins on your western blot!
Re: Western blot
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.
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