Course: SAT 170 Introductory Biotechnology

Unit 5 Learning Module

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Protein Structure and Function (continued)
Separation and Analysis: Downstream Processing (continued)

Once the protein product has been isolated from a cell, tests are performed to confirm the presence of specific protein desired. The methods used to identify the presence of a particular protein are described below.

- To identify the specific protein products a Western Blot may be performed. In this procedure, the proteins will be separated from the electrophoresis gel to a nitrocellulose membrane. Antibodies that attach to the protein of interest are then applied to the membrane. These antibodies are labeled with some type of detectable marker. If the antibody does not bind to any of the protein products, then the protein was not successfully isolated. If the antibody was able to locate the specific protein product the marker will be visible to the researcher.

See Figure 11: For a Western Blot, the proteins from an SDS Page gel must be transferred to a membrane. This also relies on an electrical current which will move the proteins from the gel onto the membrane. The antibodies can then be applied directly to the membrane for the detection of specific proteins.

Figure 11

Figure 12

Figure 13

- An Eastern Blot is similar to a Western Blot, but in this case probes or markers are used specifically to identify some of the post-translational modifications such as those discussed earlier in this unit.
- The enzyme-linked immunoassay, commonly referred to as ELISA, also relies on using antibodies for the detection of certain protein products. During this technique, a protein sample will be placed inside the wells of an ELISA plate. Thus, the bottom of the plate will be coated with a particular protein sample.  Antibodies specific to the protein of interest, or target protein are then added to the wells; these are known as primary antibodies. The wells are then washed with a buffer solution. Thus, if the primary antibody was unable to find and bind to its target protein it will be removed from the well with the buffer wash. A secondary antibody is then added to the well. This antibody will bind to the primary antibody and it is also labeled with some type of detectable marker. Once again the wells are washed with buffer. If the secondary antibody did not find and bind to the primary antibody (because the primary antibody was removed in the previous wash), then the secondary antibody will be removed with the buffer wash. Thus, if the marker is observed once the assay is complete that means that the primary antibody was able to find and bind to the target protein, which allowed the labeled secondary antibody to bind to it; therefore, the specific protein product was present.

See Figure 12: The ELISA plate has several wells to which different samples can be applied. Often, the same sample is applied to several wells at once to ensure accurate detection of a specific protein.

See Figure 13: The specific antibodies (with the green ball) bind to the protein sample which has been laid down on the bottom of the ELISA well (shown as dark black line). Once the specific antibody binds, the non-specific antibody with the detectable marker (yellow ball) will bind. The presence of the detectable marker indicates that the original protein was present.