Difference between revisions of "20.109(S14):Begin Western protein analysis (Day2)"
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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. | 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 | + | 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. | ||
− | + | 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 s immunogenic response, the antigen is often injected multiple times over several weeks in the presence of an immune-boosting compound called adjuvant. After some time, usually 4 to 8 weeks, samples of the animal s blood are collected and the cellular fraction is removed by centrifugation. What is left, called the serum, can then be tested in the lab for the presence of specific antibodies. Even the very best antisera have no more than 10% of their antibodies directed against a particular antigen. The quality of any antiserum is judged by its purity (that it has few other antibodies), its specificity (that it recognizes the antigen and not other spurious proteins) and its concentration (sometimes called its titer). Animals with strong responses to an antigen can be boosted with the antigen and then bled many times, so large volumes of antisera can be produced. However animals have limited life-spans and even the largest volumes of antiserum will eventually run out, requiring a new animal for immunization. The purity, specificity and titer of the new antiserum will likely differ from that of the first batch. High titer antisera against bacterial and viral proteins can be particularly precious since these antibodies are difficult to raise; most animals have seen these immunogens before and therefore don t mount a major immune response when immunized. Antibodies against toxic proteins are also challenging to produce if they make the animals sick. | ||
− | + | [[Image: Macintosh HD-Users-nkuldell-Desktop-monoclonalAb.png|center|425px|generating monoclonal antibodies]] | |
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− | + | Monoclonal antibodies overcome many limitations of polyclonal pools in that they are specific to a particular epitope and can be produced in unlimited quantities. However, more time is required to establish these antibody-producing cells, called hybridomas, and it is a more expensive endeavor. In this process, normal antibody-producing B cells are fused with immortalized B cells derived from myelomas, and the two cell types are fused by chemical treatment with a limited efficiency. To select only heterogeneously fused cells, the cultures are maintained in medium in which myeloma cells alone cannot survive (often HAT medium). Normal B cells will naturally die out over time with no intervention, so ultimately only the fused cells, called hybridomas, remain. A fused cell with two nuclei can be resolved into a stable cell line after mitosis. Production of stable hybridomas is tedious and difficult but often worth the effort since monoclonal antibodies can recognize covalently-modified epitopes specifically. These are invaluable for experimentally distinguishing the phosphorylated or glycosylated forms of an antigen from the unmodified forms. | |
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− | + | For Western analysis, a high quality antibody can have a relatively low affinity for its target protein. This is because the target is localized and concentrated on a blot, allowing the antibody to bind using both antibody |