Difference between revisions of "20.109(S14):Begin Western protein analysis (Day2)"
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==Introduction== | ==Introduction== | ||
− | + | Last time you got familiar with the two cell lines that we will be using during Module 2, and plated a known quantity of each cell type in preparation for protein analysis. Today you will lyse the cells, isolate the protein fraction, and separate the proteins on an acrylamide gel. The teaching faculty will probe the gels for Ku80 expression with a primary antibody, and a week from now you will complete the final steps of this assay and visualize your Western blot. | |
− | + | After completing the Day 1 exercise and hearing the Day 2 lecture, you should be well poised to understand that performing this Western blot serves a true research purpose. The Ku-80 deficient xrs6 cell line can revert to wild-type Ku80 expression under certain circumstances, and thus we want to validate that our current crop of xrs6 are not revertants. Recall that xrs6 have one XRCC5 allele that codes for truncated, non-functional Ku80, but also another, wild-type allele that is normally silenced by methylation. Should this allele become demethylated, the cells will no longer be repair deficient, and we ll have some ''really'' boring flow cytometry results! Of course, even if the xrs6 strain couldn t revert, we would want to sneak a Western into 20.109 for educational purposes. But isn t it nice to know that you can formally motivate this experiment for your research article? | |
− | + | A great way to identify a specific protein from a complex mixture is to exploit antibodies – also called immunoglobulins. We will use antibodies to perform a Western blot in this module, and to perform an enzyme-linked immunosorbent assay (ELISA) during Module 3. We'll learn more about the structure of antibodies, and different contexts in which they are used, during Module 3. For now, we'll discuss where they come from. | |
− | A great way to identify a specific protein from a complex mixture is to exploit antibodies | + | |
− | + | Many species can be used to raise antibodies. Most commonly mice, rabbits, and goats are immunized, but other animals like sheep, chickens, rats and even humans can be used. The protein used to raise an antibody is called the antigen and the portion of the antigen that is recognized by an antibody is called the epitope. Each antibody can recognize only a small portion of its antigen, typically 5 to 6 amino acids. Some antibodies are monoclonal, or more appropriately monospecific, and recognize one epitope, while other antibodies, called polyclonal antibodies, are in fact antibody pools that recognize multiple epitopes. | |
− | + | [[Image: Macintosh HD-Users-nkuldell-Desktop-polyclonalAb.png|center|425px|generating polyclonal antibodies]] | |
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+ | To raise polyclonal antibodies, the antigen of interest is first purified and then injected into an animal. To elicit and enhance the animal |