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When performing a western blot, what is the purpose of adding a secondary antibody?
Typically, the secondary antibody is designed to have either a fluorescent or colorimetric tag to allow for detection. The primary antibody binds to the protein of interest, but (usually) does not have its own tag. The protein samples have already been properly separated during electrophoresis. Noise is blocked via various methods, but not by the secondary antibody. The secondary antibody does not influence the binding of the primary antibody.
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A student researcher overexpresses an exogenous protein in cell culture and wants to determine if that protein, is in fact, overexpressed. What technique would best demonstrate that this protein is expressed in these cells?
The correct answer is Western blot. Western blots utilize antibodies to detect specific proteins in a cell lysate. Northern blots detect specific RNA within a sample, whereas Southern blots detect specfic DNA sequences within a sample. An EMSA detects whether or not a protein is active, and therefore can bind a specific sequence of DNA.
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After proteins are run on an SDS-PAGE gel, a transfer is executed. What is the purpose of the transfer in Western blot protocol?
After proteins are run on an SDS-PAGE gel and separated by size, they are transferred to a nitrocellulose membrane. This exposes the proteins so that an antibody can recognize and bind to the protein of interest. Once the antibody is bound, a fluorescent secondary conjugated antibody will facilitate the visualization of the protein of interest.
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Which of the following is not a similarity between enzyme-linked immunosorbent assays (ELISAs) and western blots, two common protein detection methods?
The major difference between ELISA and western blot is the fact that ELISA detects naive protein in its original conformation, while western requires denaturation of the protein by SDS-PAGE prior to detection. This makes western blotting more stringent and better for quantification, although both assays can quantitatively assess protein levels if done properly.
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