GRE Subject Test: Biology : Understanding Western Blots

Study concepts, example questions & explanations for GRE Subject Test: Biology

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Example Questions

Example Question #6 : Lab Techniques

When performing a western blot, what is the purpose of adding a secondary antibody?

Possible Answers:

Allow for detection of the protein sample

Block any interfering noise coming from the membrane

Ensure that the primary antibody binds properly to the sample

Separate the sample from other proteins

Correct answer:

Allow for detection of the protein sample

Explanation:

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. 

Example Question #1 : Understanding Western Blots

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?

Possible Answers:

Electrophoretic mobility shift assay (EMSA)

None of the other answers

Western blot 

Northern blot

Southern blot

Correct answer:

Western blot 

Explanation:

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.  

Example Question #8 : Lab Techniques

After proteins are run on an SDS-PAGE gel, a transfer is executed. What is the purpose of the transfer in Western blot protocol?

Possible Answers:

Probe the gel with an antibody to detect a protein of interest

Denature the proteins in the sample 

Visualize the proteins run on the gel 

Move proteins from the SDS-PAGE gel to a nitrocellulose membrane

None of the other anwers

Correct answer:

Move proteins from the SDS-PAGE gel to a nitrocellulose membrane

Explanation:

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. 

Example Question #9 : Lab Techniques

Which of the following is not a similarity between enzyme-linked immunosorbent assays (ELISAs) and western blots, two common protein detection methods?

Possible Answers:

Tissue sample must be homogenized and the protein extracted to utilize the assay

Requirement of antibodies conjugated to a marker for detection

Requirement that the protein is denatured prior to detection

Detection of protein using antibodies specifically generated against antigens

Information from the assays can be made quantitative with the right controls

Correct answer:

Requirement that the protein is denatured prior to detection

Explanation:

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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