However, more time is required to establish these antibody-producing cells, called hybridomas, and it is a more expensive endeavor. Monoclonal antibodies overcome many limitations of polyclonal pools in that they are specific to a particular epitope and can be produced in unlimited quantities. Antibodies against toxic proteins are also challenging to produce if they make the animals sick. 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. The purity, specificity and titer of the new antiserum will likely differ from that of the first batch. However animals have limited life-spans and even the largest volumes of antiserum will eventually run out, requiring a new animal for immunization. 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. 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). Even the very best antisera have no more than 10% of their antibodies directed against a particular antigen. What is left, called the serum, can then be tested in the lab for the presence of specific antibodies. After some time, usually 4 to 8 weeks, samples of the animal’s blood are collected and the cellular fraction is removed by centrifugation.
![kaleidoscope protein ladder kaleidoscope protein ladder](https://www.licor.com/bio/images/figures/reagents/chameleon-duo-image.png)
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. To raise polyclonal antibodies, the antigen of interest is first purified and then injected into an animal. 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. Each antibody can recognize only a small portion of its antigen, typically 5 to 6 amino acids. 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.
![kaleidoscope protein ladder kaleidoscope protein ladder](https://www.mdpi.com/ijms/ijms-18-00089/article_deploy/html/images/ijms-18-00089-g004.png)
Most commonly mice, rabbits, and goats are immunized, but other animals like sheep, chickens, rats and even humans can be used. Many species can be used to raise antibodies. For now, we'll discuss where they come from. We'll learn more about the structure of antibodies, and different contexts in which they are used, during Module 3.
![kaleidoscope protein ladder kaleidoscope protein ladder](https://82879-229730-raikfcquaxqncofqfm.stackpathdns.com/wp-content/uploads/2015/04/PM001-0500-BLUltra-Prestained-Protein-Standard.jpg)
We will use antibodies to perform a Western blot in this module, and to perform an enzyme-linked immunosorbent assay (ELISA) during Module 3. But isn’t it nice to know that you can formally motivate this experiment for your research article?Ī great way to identify a specific protein from a complex mixture is to exploit antibodies – also called immunoglobulins. 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. 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. 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. 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.Īfter 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. Today you will lyse the cells, isolate the protein fraction, and separate the proteins on an acrylamide gel. 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. 2.4 Part 4: Transfer proteins to membrane.2.3 Part 3: Separate proteins by SDS-PAGE.