Molecular biologists undertake a series of experiments designed to classify proteins involved in various intracellular signaling pathways. During one of the experiments, a protein mixture obtained from a cell culture is separated by gel electrophoresis and subsequently transferred to a filter membrane. Labeled double-stranded DNA probes are then used to detect a specific protein of interest in the sample. Which of the following proteins is most likely to be detected by this method?
Blotting technique Substance detected Type of probe Northern RNA Single-stranded DNA or RNA (hybridization probe) Southern DNA Western Protein Antibody Southwestern DNA-binding protein Double-stranded DNA
The Southern, Western, Northern, and Southwestern blot procedures are powerful techniques used to analyze and identify DNA fragments, proteins, mRNA, and DNA-bound proteins, respectively. The same basic technique underlies all of the blot procedures. First, the unknown sample is separated by gel electrophoresis. Separation occurs based on a molecule's size and charge. The separated molecules form bands on the gel that are then blotted onto a nitrocellulose membrane and incubated with a labeled probe to identify the specific DNA fragment, RNA molecule, or protein of interest.
Southwestern blots are used to identify and isolate proteins that bind DNA. In this technique, the target protein binds to a labeled, double-stranded DNA probe that is homologous to the protein’s regulatory sequence. Of the molecules listed, c-Jun is the only DNA-binding protein. c-Jun and c-Fos are nuclear transcription factors that directly bind DNA via a leucine zipper motif. The genes that code for c-Jun and c-Fos are proto-oncogenes, genes that can become oncogenes following a mutation or with constitutive expression.
(Choice A) Ras is a proto-oncogene that codes for a membrane-bound G-protein. This G-protein acts as a secondary mediator for several hormones and cytokines that act on cell membrane receptors. Ras activation activates the MAP kinase pathway and ultimately affects transcription. However, Ras itself does not bind directly to DNA.
(Choices C and E) The β1-adrenergic receptor is a classic Gs-protein-coupled receptor located in the cell membrane. It does not interact directly with DNA. Adenylyl cyclase is the enzyme that cleaves ATP to form cAMP, the second messenger associated with Gs-protein-coupled receptors. cAMP activates protein kinase A for further downstream signaling.
(Choice D) S-100 proteins are homodimeric calcium-binding proteins, similar in structure to calmodulin and important in intracellular functions such as protein phosphorylation and cell growth and differentiation. S-100 is a marker for cells of neural crest derivation (melanocytes and Schwann cells), as well as Langerhans cells and other dendritic cells.
Educational objective:
Southwestern blotting is used to detect DNA-binding proteins such as transcription factors, nucleases, and histones.