> See also: > - [[Antibodies]] # Antibody-Based Lab Techniques ## Western Blot > Southern (Edwin Southern) -> DNA > Northern -> RNA > Western -> Protein **Western blotting** (also known as *immunoblotting*) is a technique that allows researchers to identify a specific immobilized protein using an antigen specific antibody ### Western Transfer After a gel is run, it can be "imprinted" onto a special paper known as a **nitrocellulose membrane** - Its important to begin a transfer as soon as a gel is complete, as the samples can begin to diffuse through the gel matrix and cause the bands to become "puffier" if left sitting for too long (~20 min is too long) - After the transfer has been completed, the nitrocellulose membrane can be stored for long periods of time (over a year) Once a western **Nanobodies** are small, single-domain antibodies ## Enzyme-Linked Immunosorbent Assay (ELISA) > [!note] QBM Objectives > **Chapter 11: Western blot, ELISA, and Immunoprecipitation** - [ ] *Western Blot Basics* - [ ] Understand the basics of using western blots to confirm the presence of a protein following SDS-PAGE. - [ ] *Antibodies* - [ ] Define specificity, sensitivity, and affinity. - [ ] Compare and contrast the advantages and disadvantages of polyclonal vs. monoclonal antibodies. - [ ] *Western Blot Procedure* - [ ] Describe the steps of western blotting. - [ ] *Western Blot Transfer Membrane and Blocking* - [ ] Compare and contrast nitrocellulose and PVDF membranes. - [ ] Describe the importance of the blocking step in western blotting. - [ ] *Primary Antibodies* - [ ] Recall how to choose a primary antibody, including which species to use and what kind of epitope should be recognized (linear vs. conformational). - [ ] *Secondary Antibodies* - [ ] Choose a secondary antibody given a primary antibody. - [ ] Describe the advantages of using a primary/secondary antibody system over a conjugated primary antibody. - [ ] *Western Blot Analysis* - [ ] Given an SDS-PAGE and western blot, determine the number of different antibodies needed to generate the western blot. Recall that only one secondary is needed for all of the primaries. - [ ] *ELISA* - [ ] Describe the Enzyme-Linked Immunosorbent Assay (ELISA), recalling specific examples of when it is used. - [ ] Define and differentiate between direct, indirect, sandwich, competitive, and reverse ELISAs. - [ ] Understand why ELISA HIV tests can produce false positives, and how it can be verified. - [ ] *ELISA Identification* - [ ] Recognize examples of the different ELISA types. - [ ] *IP, Co-IP, RIP* - [ ] Define immunoprecipitation, co-immunoprecipitation, RNA immunoprecipitation, and chromatin immunoprecipitation.