Structural Biochemistry/Identifying Proteins
After purification is complete, how will you prove that you have successfully isolated the correct protein? Several techniques can be used to identify whether or not the isolated protein is the desired one, including immunological reactions.
Overview Millions of antibodies are produced by the body, with each one tailored to recognize specific protein structures. The "Y" shaped antibody recognizes protein structures through its binding site, which is able to attach to antigens with the perfect fit by forming intermolecular bonds. After being exposed to a pathogen, organisms can churn out several different antibodies that will recognize this same pathogen for every subsequent exposure. These polyclonal antibodies attach to different areas on the same pathogen to counteract mutations that change a pathogen's surface proteins and render a specific antibody recognition site obsolete.
Monoclonal Antibodies Though useful from an organism's standpoint, polyclonal antibodies prove to be messy and inefficient in the lab because the body does not produce them in exact ratios. Different antibody samples would consist of different relative amounts of several antibodies, each of which attach differently to the protein product. So how can a researcher force a model organism to create only one type of antibody for a particular protein? The solution was discovered by Cesar Milstein and Georges Köhler, who mixed anti-body producing cells with immortal cancer cells (Meyloma cells) capable of mass producing identical proteins over and over again. The hybrid cells capable of producing the desired antibody could then be selected and grown in mass culture or within the model organism itself as tumors.
Enzyme-linked Immunosorbent Assay (ELISA) There are two types of ELISA, "Indirect" and "Sandwich." Both use a specific antibody to recognize the desired protein. This first antibody must be specially produced for each and every different protein. After unbound antibodies or proteins are washed away, a second antibody that contains an enzyme capable of producing a visual confirmation that the isolated protein is present is introduced to solution. This second antibody is a generic antibody that can be used regardless of the specific protein.
Indirect ELISA: 1) A container is coated with protein.2)The first antigen, specific to the protein, binds to the protein. 3)The container is washed. If the desired protein is not present, the antibodies will not bind and will be removed from solution. 4)The second antibody with an enzyme is added and binds to the first antibody. 5)Binding to the first antibody induces a chemical reaction that causes a visually identifiable change in solution (color change or fluorescence), indicating that the first antibody is present, which in turn indicates that the desired protein is also present. SEE FIGURE 1.
Sandwich ELISA: 1) A container is coated with the monoclonal antibody. 2)The protein is added and will bind to the antibody only if it is the desired protein. 3) The container is washed. Only the desired protein and antigens will remain (if any). 4) A second antibody linked to an enzyme is added and will attach to the protein. 5) Attaching to the protein will induce a chemical change that allows for visual confirmation that the protein is present. Note that since the second enzyme is attaching directly to the protein, the rate of visual change can be used to determine the amount of protein present. SEE FIGURE 2
Western Blotting 1) After separating the desired protein from other proteins or molecular impurities via gel electrophoresis, the resulting protein bands are transferred from the gel to a thin polymer sheet. This makes the proteins more accessible to reactions. 2) The monoclonal antibody is added. Only the desired protein will react with the antibody, so only one band will have antibodies attached. 3) The polymer sheet is washed to remove unbound antibodies. 4)A second antibody linked to an enzyme attaches to the first. 5) A chemical reaction induces a visual change in the band containing the desired antibody. Or photographic film can overlay the sheet and record the protein band that contains the attached antibodies. SEE FIGURE 3.