There are 3 principal stages of this technique that are essential for a quality outcome: Electroblotting, Immunoblotting, and Detection. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. These signals can then be imaged and quantified using a process called densitometry. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. Next, the membranes are probed with antibodies in a process called immunboblotting. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |