GRE Subject Test: Biochemistry, Cell, and Molecular Biology : Help with Separation Techniques

Study concepts, example questions & explanations for GRE Subject Test: Biochemistry, Cell, and Molecular Biology

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All GRE Subject Test: Biochemistry, Cell, and Molecular Biology Resources

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Example Questions

Example Question #1 : Help With Separation Techniques

Which of the following techniques would be most useful to isolate intact ribosomes from cells?

Possible Answers:

Centrifugation

SDS-PAGE

Dialysis

Western blotting

Correct answer:

Centrifugation

Explanation:

Ribosomes have relatively small molecular weights in comparison to cell organelles, and relatively large molecular weights in comparison to macromolecules (such as proteins). Several rounds of centrifugation, using slightly different conditions, is a feasible way to isolate intact ribosomes.

SDS-PAGE is an excellent technique for separating denatured proteins based on molecular weight. Western blotting is used to detect the presence specific proteins in a sample. Dialysis is often used in protein purification procedures.

Example Question #2 : Help With Separation Techniques

__________ is a technique used to pull a protein complex out of a solution by targeting one member of the complex with an antibody. 

Possible Answers:

Mass spectrometry 

FRET

Southern blotting

Co-immunoprecipitation

Correct answer:

Co-immunoprecipitation

Explanation:

The only answer that will viably pull a protein complex out of a solution is co-immunoprecipitation. Co-immunoprecipitation involves using a large amount of antibody for a specific protein to capture the protein of interest and anything that is associated with it (namely the protein complex). The proteins can then be eluted from the antibody and analyzed.

FRET involves fluorescently labeling two proteins to look for, amongst other possible uses, protein-protein interactions in vivo. Southern blotting is a technique used to separate and identify a specific DNA sequence in a solution. Mass spectrometry can be used to analyze and sequence proteins, but does not directly involve the use of antibodies.

Example Question #3 : Help With Separation Techniques

Ethidium bromide is commonly used to detect presence of DNA in DNA gels under UV light. What type of bond does ethidium bromide form with double-stranded DNA to emit fluorescence?

Possible Answers:

Covalent bond

Metallic bond

Van der Waals bond

Ionic bond

Correct answer:

Van der Waals bond

Explanation:

Ethidium bromide intercalates into the hydrophobic interior of the DNA double helix and remains bound through Van der Waals interactions. The binding of ethidium bromide to DNA leads to a conformational shift that increases its fluorescence.

Example Question #4 : Help With Separation Techniques

A new RNA virus was identified, and the high-throughput sequencing of the genome resulted in two unique contigs. Which method would provide insight into the genome structure of this virus?

Possible Answers:

A Northern blot analysis using probes from the sequenced regions

A denaturing protein gel to visualize all proteins in the viral particles

A PCR with primers against the sequenced regions

A Southern blot analysis using probes based on closely related virus

Correct answer:

A Northern blot analysis using probes from the sequenced regions

Explanation:

Obtaining two distinct contigs from a sequencing reaction suggests that either this virus has two separate chromosomes, or that there is some type of error in the sequencing reaction. 

The most direct way to test this would be to run a Northern blot analysis with probes against the two potential chromosomes. If they are unique chromosomes, we would expect to see a band that matches the length of the sequenced product on the gel per chromosome. If it is an error, both probes should yield a band of the same size, suggesting that there is only one chromosome.

A Southern blot tests for the presence of DNA; therefore, that would not be useful. A protein gel would also not be useful in this case since we are trying to determine the presence of RNA. A PCR will also not be fruitful since it cannot be conducted on RNA.

Note that a reverse-transcription PCR could potentially be useful because it would convert the RNA into DNA; in that case, you can design mulitple combinations of primers if you think it is a single chromosome.

Example Question #5 : Help With Separation Techniques

Which of these options is an important step in the process of DNA extraction and isolation?

Possible Answers:

Removal of proteins from sample

Homogenization of cells or tissues

Precipitation of DNA with an alcohol

All of these

Removal of contaminants by an organic solvent

Correct answer:

All of these

Explanation:

These are all important steps to create a usable DNA extraction. First, the tissues are homogenized, generally chemically or mechanically. The proteins are then removed from the sample by denaturation. Any contaminants can be removed by multiple methods with organic solvents. Depending on the situation, some other steps are involved, such as removal of RNA from the extract. Lastly, the DNA is precipitated via an alcohol into a pure DNA extract.

Example Question #6 : Help With Separation Techniques

What is the main denaturing agent in a western blot gel that disrupts secondary, tertiary, and quaternary protein structure? 

Possible Answers:

Acrylamide 

Tetramethylethylenediamine (TEMED)

Ethylenediaminetetraacetic acid (EDTA)

Sodium dodecyl sulfate (SDS)

Ammonium persulfate

Correct answer:

Sodium dodecyl sulfate (SDS)

Explanation:

The correct answer is SDS. SDS disrupts non-covalent bonds in proteins ultimately denaturing these proteins. Both ammonium persulfate and TEMED are reagents used to polymerize (or solidify) SDS-page gels for western blot applications. EDTA chelates cations in solution preventing the activity of proteases that may degrade the primary amino acid structure. Acrylamide is the substance that is polymerized in the SDS-gel, giving the viscous physical characteristics to the gel. 

Example Question #7 : Help With Separation Techniques

White blood cells and red bloods cells differ in numerous physical properties, one of which is a significant difference in the density of the two cell types. Red blood cells do not have nuclei, making them much less dense and easy to separate out from white blood cells through simple centrifugation techniques. However, there are numerous white blood cell subtypes with similar densities, presenting a challenge in studying specific white blood cell types as they are more difficult to separate out. We can take advantage of the fact that different white blood cell types express different cell-surface molecules.

Which of the following techniques would be most useful to utilize different cell-surface molecules to separate out different types of white blood cells?

Possible Answers:

Southern blotting

Flow cytometry 

Centrifugation 

Co-immunoprecipitation 

X-ray crystallography 

Correct answer:

Flow cytometry 

Explanation:

Flow cytometry utilizes antibodies specific to a protein expressed by a single cell type. Antibodies can be coated onto magnetic beads, and those antibodies will bind only cells with the proper proteins, thus becoming "stuck" to the beads which can be separated out from the sample. Centrifugation will not be able to detect such small differences, and is only useful if the density is different. Co-IP is more appropriate when looking for proteins that interact or colocalize. Southern blotting is for DNA detection, and X-ray crystallography is for understanding protein structure. 

All GRE Subject Test: Biochemistry, Cell, and Molecular Biology Resources

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