GRE Subject Test: Biology › Electrophoresis and Blots
Which of the following is a primary factor that dictates how far a protein will migrate during SDS-PAGE?
Size
Degree of secondary structure
Degree of tertiary structure
Number of subunits
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.
Which of the following is a primary factor that dictates how far a protein will migrate during SDS-PAGE?
Size
Degree of secondary structure
Degree of tertiary structure
Number of subunits
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.
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?
Western blot
Electrophoretic mobility shift assay (EMSA)
Southern blot
Northern blot
None of the other answers
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.
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?
Western blot
Electrophoretic mobility shift assay (EMSA)
Southern blot
Northern blot
None of the other answers
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.
A cell line was engineered to contain and express a DNA sequence frequently associated with pancreatic cancer, with the hope that the cell line will undergo rapid cell division much like the cancerous cells. You want to test whether or not the cell line is efficiently transcribing this DNA by measuring the quantity of mRNA produced. Which of the following methods would be best suited for testing this?
Northern blot
Southern blot
Western blot
Thin-layer chromatography
Two-photon microscopy
Northern blots are designed to detect the quantity and identity of mRNA transcripts in cells or tissue. Electrophoresis separates RNAs by size, and then hybridization is used to detect the mRNA sequence of interest. Southern blots detect DNA sequences, but give no information about whether they are transcribed or not. Western blots detect proteins, but we are not interested in translation. Neither thin-layer chromatography nor two-photon microscopy would tell us anything about the genetic processes occurring in the cell line.
A cell line was engineered to contain and express a DNA sequence frequently associated with pancreatic cancer, with the hope that the cell line will undergo rapid cell division much like the cancerous cells. You want to test whether or not the cell line is efficiently transcribing this DNA by measuring the quantity of mRNA produced. Which of the following methods would be best suited for testing this?
Northern blot
Southern blot
Western blot
Thin-layer chromatography
Two-photon microscopy
Northern blots are designed to detect the quantity and identity of mRNA transcripts in cells or tissue. Electrophoresis separates RNAs by size, and then hybridization is used to detect the mRNA sequence of interest. Southern blots detect DNA sequences, but give no information about whether they are transcribed or not. Western blots detect proteins, but we are not interested in translation. Neither thin-layer chromatography nor two-photon microscopy would tell us anything about the genetic processes occurring in the cell line.
As opposed to electrophoresis with a more standard agar gel, what does polyacrylamide gel electrophoresis (PAGE) allow for when working with DNA?
Resolution down to DNA strands with single base length differences
Running multiple DNA strands in a single gel lane
Staining the DNA for visualization
Running multiple lanes in one gel
None of these
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).
As opposed to electrophoresis with a more standard agar gel, what does polyacrylamide gel electrophoresis (PAGE) allow for when working with DNA?
Resolution down to DNA strands with single base length differences
Running multiple DNA strands in a single gel lane
Staining the DNA for visualization
Running multiple lanes in one gel
None of these
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).
When performing a western blot, what is the purpose of adding a secondary antibody?
Allow for detection of the protein sample
Ensure that the primary antibody binds properly to the sample
Separate the sample from other proteins
Block any interfering noise coming from the membrane
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.
When performing a western blot, what is the purpose of adding a secondary antibody?
Allow for detection of the protein sample
Ensure that the primary antibody binds properly to the sample
Separate the sample from other proteins
Block any interfering noise coming from the membrane
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.