Electrophoresis and Blots - GRE Subject Test: Biology

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.

A Northern blot is very similar to a Southern blot, except it is used to identify RNA fragments rather than DNA fragments. A gel is used to separate the RNA fragments based on size. A radioactive probe can then be used to identify which known RNA sequences have been combined with an RNA fragment.

A Western blot is used to identify protein subunits and fragments. Eastern blots are used to identify protein modifications, such as glycosylation.

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.

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.

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.

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.

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).

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.

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.

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.

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.