Western Blotting Technique Laboratory Analysis
Abstract
This experiment followed an exact protocol from EDVOTEK® Kit #17. This kit was designed to use western blot analysis to detect proteins from Non-fat powdered milk. The experiment had to be run two times before the expected outcome was reached. However, after the second attempt, it was successful. The gel ran well, and the proteins from the gel were successfully transferred to the membrane provided in the kit.
Introduction
The name western blot was given to the technique by W. Neal Burnette and is a pun on the name Southern blot. Southern blot was a name given by Edwin Southern to a technique he developed for DNA detection. Western blot analysis has the ability to detect one specific protein in a mixture of any number of proteins while at the same time giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. However, this method does require the use of a high-quality antibody directed against a desired protein. During western blotting procedures, generally, proteins are solubilized with detergents and reducing agents, and then are separated in polyacrylamide gels. After the separation, the pattern of proteins from the gel is then transferred to a nitrocellulose filter or polyvinylidene fluoride membrane. The proteins then become covalently bound to the support. Western blots and Southern are distinguished from one another by the probe. In western blots, specific unlabeled antibodies, which are typically specific to each individual protein and will therefore react specifically with the target protein. This is what allows for the recognition of the protein of interest from within the background of other cellular proteins. The bound antibody is then detected on the blot by a secondary reagent. This reagent can be either radiolabeled or coupled to an enzyme. Western blots can be used to search for the presence of certain proteins in specific samples, tissues, or
This experiment followed an exact protocol from EDVOTEK® Kit #17. This kit was designed to use western blot analysis to detect proteins from Non-fat powdered milk. The experiment had to be run two times before the expected outcome was reached. However, after the second attempt, it was successful. The gel ran well, and the proteins from the gel were successfully transferred to the membrane provided in the kit.
Introduction
The name western blot was given to the technique by W. Neal Burnette and is a pun on the name Southern blot. Southern blot was a name given by Edwin Southern to a technique he developed for DNA detection. Western blot analysis has the ability to detect one specific protein in a mixture of any number of proteins while at the same time giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. However, this method does require the use of a high-quality antibody directed against a desired protein. During western blotting procedures, generally, proteins are solubilized with detergents and reducing agents, and then are separated in polyacrylamide gels. After the separation, the pattern of proteins from the gel is then transferred to a nitrocellulose filter or polyvinylidene fluoride membrane. The proteins then become covalently bound to the support. Western blots and Southern are distinguished from one another by the probe. In western blots, specific unlabeled antibodies, which are typically specific to each individual protein and will therefore react specifically with the target protein. This is what allows for the recognition of the protein of interest from within the background of other cellular proteins. The bound antibody is then detected on the blot by a secondary reagent. This reagent can be either radiolabeled or coupled to an enzyme. Western blots can be used to search for the presence of certain proteins in specific samples, tissues, or