GRE Subject Test: Biology : Electrophoresis and Blots

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

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Example Questions

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Example Question #1 : Electrophoresis And Blots

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

Possible Answers:

Separate the sample from other proteins

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

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 : Electrophoresis And 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:

Western blot 

None of the other answers

Northern blot

Southern blot

Electrophoretic mobility shift assay (EMSA)

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 #1 : Understanding Western Blots

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:

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

Visualize the proteins run on the gel 

Denature the proteins in the sample 

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

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 #1 : Electrophoresis And Blots

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

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

Requirement that the protein is denatured prior to detection

Detection of protein using antibodies specifically generated against antigens

Requirement of antibodies conjugated to a marker for detection

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. 

Example Question #2 : Electrophoresis And Blots

A researcher is working with a protein that contains four subunits of differing molecular weights. If the researcher performs SDS-PAGE, how many distinct bands should he see on the gel?

Possible Answers:

One

Four

Two

Three

Correct answer:

Four

Explanation:

SDS-PAGE requires that proteins are denatured before they are run through the gel, typically by the addition of detergents and then heating the sample. Since the protein has four subunits that are all different molecular weights, we would see four distinct bands that represent the four subunits. If the subunits had the same molecular weight, we would only see one band.

Example Question #1 : Electrophoresis And Blots

Which of the following is a primary factor that dictates how far a protein will migrate during SDS-PAGE?

Possible Answers:

Degree of tertiary structure

Size

Number of subunits

Degree of secondary structure

Correct answer:

Size

Explanation:

The primary factor dictating how far a protein will migrate during SDS-PAGE is the size of the protein. One of the key features of SDS-PAGE is that the protein sample is denatured and covered in a detergent prior to being run through the gel. This essentially eliminates any complications from the degree of folding or the number of subunits. In fact, subunits will migrate according to their own molecular weights.

Example Question #1 : Understanding Page And Sds Page

Which of the following is true about SDS-PAGE?

Possible Answers:

Staining with ethidium bromide allows visualization of results

It separates proteins by charge

The main ingredient in the gel is agarose


It is used to anaylze DNA fragments

It requires a protein-denaturing gel

Correct answer:

It requires a protein-denaturing gel

Explanation:

SDS-PAGE requires a denaturing protein gel that separates proteins based on size. The primary ingredients are polyacrylamide and sodium dodecyl sulfate—SDS-PAGE refers to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In order to visualize the results, proteins separated via SDS-PAGE are transferred to a nitrocellulose membrane, where they are probed with antibodies for a specific protein of interest. 

Example Question #3 : Electrophoresis And Blots

As opposed to electrophoresis with a more standard agar gel, what does polyacrylamide gel electrophoresis (PAGE) allow for when working with DNA?

Possible Answers:

Running multiple lanes in one gel

Resolution down to DNA strands with single base length differences

Running multiple DNA strands in a single gel lane

None of these

Staining the DNA for visualization

Correct answer:

Resolution down to DNA strands with single base length differences

Explanation:

Polyacrylamide gels allow for resolution of DNA strands down to single base pair differences. All these other options are applicable to any kind of gel electrophoresis. This property of polyacrylamide allowed for some of the earliest forms of Sanger sequencing, in which DNA sequences were read by their respective chain lengths across a 4 column gel (with one column each for adenine, cytosine, guanine, and thymine).

Example Question #1 : Electrophoresis And Blots

Which of the following techniques would be most useful to study gene expression?

Possible Answers:

Northern blot

Southern blot

Eastern blot

Western blot

Correct answer:

Northern blot

Explanation:

In order to study gene expression it would be useful to quantify the expression of a specific RNA transcript. All of the blotting techniques follow similar procedures, but differ in the macromolecule they separate or the information they obtain. Southern blotting is used for DNA fragments, western blotting is used for proteins, and eastern blotting is used to look at post-translational modifications. Northern blotting would be the most useful in this question, as it is used to look at RNA fragments.

Example Question #1 : Electrophoresis And Blots

Which of the following probes are most commonly used in southern blotting?

Possible Answers:

Antibodies

Nucleic acids

Phosphorous-32

Biotin-binding proteins

Correct answer:

Nucleic acids

Explanation:

Southern blotting is a technique used to detect a specific DNA sequence in a sample. The sample is run on an agarose gel and transferred to a membrane. A labeled nucleic acid probe is then incubated with the membrane. The probe carries a tag for identification and will only bind to the target region of DNA. If the tag is identified, then the researcher can conclude that the target gene is present in the sample.

Antibodies are most commonly used to detect specific proteins in western blotting. Biotin-binding proteins have wide uses in both detection and purification procedures. Phosphorous-32 is commonly used to label nucleotides (such as GTP) to perform enzymatic assays.

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