Help with Other Laboratory Techniques - GRE Subject Test: Biochemistry, Cell, and Molecular Biology

Sodium fluoride is a serine/threonine phosphatase inhibitor. This is important to have in a cell lysis buffer if the protein of interest is phosphorylated. The presence of sodium fluoride will prevent the phosphoryl group from being removed by a serine/threonine phosphatase before it can be studied. It is often added to the cell lysis buffer along with sodium vanadate, which is a tyrosine/alkaline phosphatase inhibitor.

The correct answer is electrophoretic mobility shift assay (EMSA). In an EMSA, specific DNA sequences are radioactively labelled and incubated with either protein lysates or purified proteins. The protein-DNA mixture is then loaded onto a non-denaturing gel to separate the proteins and DNA by size. DNA that is bound by protein will migrate more slowly than unbound DNA, which will migrate quickly. To detect radioactivity, a film is exposed to the gel, developed and interpreted. It is important to remember that the radioactivity is emitted by the DNA and not the protein.

The correct answer is Western blot. In a Western blot, cell protein lysates are run on a denaturing gel and probed with a specific antibody. The student researchers can detect the existence of the DNA polymerase protein in the cell lysate using a specific antibody to that polymerase.

EMSAs determine a protein's ability to bind a DNA sequence, while electroporation, PCR, and restriction digests are not methods to detect protein expression.

The correct answer is DNA microarray. In a DNA microarray, short DNA sequences (probes) are labelled on a microarray chip. Probes for the same gene are clustered in the same area on the array. cDNA from a sample is labelled with a fluorescent dye and washed over the chip. If a certain gene is more highly expressed, its cDNA will bind the respective probes and give a higher intensity than a lower expressed gene.

The correct answer is whether the C-terminus of the protein is extracellular or intracellular. This is a very common laboratory technique to determine which terminus of a transmembrane protein is intracellular and extracellular. Since the antibody is specific to the C-terminus of the protein and if the C-terminus of the protein is intracellularly localized, we will only be able to detect the protein if the cells are permeabilized. In cells that are not permeabilized, the antibody will not be able to enter the cells, but permeabilization will allow the antibody to pass through the plasma membrane and bind the C-terminus. If the C-terminus is extracellular, the antibody will bind regardless of permeabilization.

All of the given answers are valid methods to introduce exogenous DNA into a cell. When working with bacteria, transformations are a simple way to introduce plasmid DNA into the cell by chemical competency. Electroporation electrocutes both bacterial and cell culture cells to introduce exogenous DNA. Infection of target cells may also be a viable method if the laboratory is equipped to generate live virus containing exogenous DNA to be introduced. Finally, transfection refers to a process mainly reserved for cell culture that introduces exogenous DNA through liposome fusion (containing DNA) with cell membranes.

Microarrays allow for the study of expression levels of thousands of genes simultaneously. Although microarrays do not tell you anything about the actual sequence of the genes, they are the only option for studying the expression levels of massive numbers of genes. It is possible to study expression levels using PCR, but not on a large scale.

Running allozyme gels was one of the first methods used in molecular studies of these enzymes that have slightly different functions due to having slightly different genetic codes (alleles). This term refers specifically to enzymatic variation created by genetic variation/alleles.

The correct answer is to separate proteins and other impurities from DNA or RNA. An extraction by phenol followed by chloroform will separate proteins and other impurities from DNA and RNA (in suspended fraction). Next, depending on the type and concentration of salt used, a researcher can selectively precipitate either DNA or RNA from the mixture with the addition of ethanol. This is a simple method to obtain pure clean ribonucleic acids from impure sources.

The correct answer is lysis of the plasma membrane. While detergents such as sodium dodecyl sulfate are commonly used in western blots as a protein denaturing agent, it also breaks down the plasma membrane by emulsifying membrane-bound lipids and proteins.

Sodium fluoride is a serine/threonine phosphatase inhibitor. This is important to have in a cell lysis buffer if the protein of interest is phosphorylated. The presence of sodium fluoride will prevent the phosphoryl group from being removed by a serine/threonine phosphatase before it can be studied. It is often added to the cell lysis buffer along with sodium vanadate, which is a tyrosine/alkaline phosphatase inhibitor.

The correct answer is electrophoretic mobility shift assay (EMSA). In an EMSA, specific DNA sequences are radioactively labelled and incubated with either protein lysates or purified proteins. The protein-DNA mixture is then loaded onto a non-denaturing gel to separate the proteins and DNA by size. DNA that is bound by protein will migrate more slowly than unbound DNA, which will migrate quickly. To detect radioactivity, a film is exposed to the gel, developed and interpreted. It is important to remember that the radioactivity is emitted by the DNA and not the protein.

The correct answer is Western blot. In a Western blot, cell protein lysates are run on a denaturing gel and probed with a specific antibody. The student researchers can detect the existence of the DNA polymerase protein in the cell lysate using a specific antibody to that polymerase.

EMSAs determine a protein's ability to bind a DNA sequence, while electroporation, PCR, and restriction digests are not methods to detect protein expression.

The correct answer is DNA microarray. In a DNA microarray, short DNA sequences (probes) are labelled on a microarray chip. Probes for the same gene are clustered in the same area on the array. cDNA from a sample is labelled with a fluorescent dye and washed over the chip. If a certain gene is more highly expressed, its cDNA will bind the respective probes and give a higher intensity than a lower expressed gene.

The correct answer is whether the C-terminus of the protein is extracellular or intracellular. This is a very common laboratory technique to determine which terminus of a transmembrane protein is intracellular and extracellular. Since the antibody is specific to the C-terminus of the protein and if the C-terminus of the protein is intracellularly localized, we will only be able to detect the protein if the cells are permeabilized. In cells that are not permeabilized, the antibody will not be able to enter the cells, but permeabilization will allow the antibody to pass through the plasma membrane and bind the C-terminus. If the C-terminus is extracellular, the antibody will bind regardless of permeabilization.

All of the given answers are valid methods to introduce exogenous DNA into a cell. When working with bacteria, transformations are a simple way to introduce plasmid DNA into the cell by chemical competency. Electroporation electrocutes both bacterial and cell culture cells to introduce exogenous DNA. Infection of target cells may also be a viable method if the laboratory is equipped to generate live virus containing exogenous DNA to be introduced. Finally, transfection refers to a process mainly reserved for cell culture that introduces exogenous DNA through liposome fusion (containing DNA) with cell membranes.

Microarrays allow for the study of expression levels of thousands of genes simultaneously. Although microarrays do not tell you anything about the actual sequence of the genes, they are the only option for studying the expression levels of massive numbers of genes. It is possible to study expression levels using PCR, but not on a large scale.

Running allozyme gels was one of the first methods used in molecular studies of these enzymes that have slightly different functions due to having slightly different genetic codes (alleles). This term refers specifically to enzymatic variation created by genetic variation/alleles.

The correct answer is to separate proteins and other impurities from DNA or RNA. An extraction by phenol followed by chloroform will separate proteins and other impurities from DNA and RNA (in suspended fraction). Next, depending on the type and concentration of salt used, a researcher can selectively precipitate either DNA or RNA from the mixture with the addition of ethanol. This is a simple method to obtain pure clean ribonucleic acids from impure sources.

The correct answer is lysis of the plasma membrane. While detergents such as sodium dodecyl sulfate are commonly used in western blots as a protein denaturing agent, it also breaks down the plasma membrane by emulsifying membrane-bound lipids and proteins.