Question

Suppose you are working on a set of experiments using Western blotting. Using an antibody that recognizes amino sequences encoded by exon 3 of a five-exon gene, you have demonstrated that a protein is highly expressed in a particular cell line. To assess the mRNA, you design reverse transcriptase polymerase chain reaction (PCR) primers to exons 2 and 4, which flank exon 3. But despite all your efforts, you never detect an mRNA. You know that it is indeed expressed, since the protein is detectable. You also know that your "positive controls" are working and your experimental procedure is sound, since the use of other cell lines allows you to readily detect an mRNA. Given what you know about the "central dogma" and gene structure, how might you explain the inability to identify the mRNA encoding this protein?

Answer 1

Western blotting may be defined as the technique used to determine the specific proteins from the given sample. This technique is also known as protein immunoblot.

The cell contains five exons, the cell line is not able to express exons 2, 4 and both. The loss of even one exon do not allow the PCR amplification. The cell line may express the mRNA that are able to use the exons 5, 3 and 1. This case allow the protein detection by western blot because the antibody has the ability to recognize the protein sequence at the exon 3. The primers has been designed by hybridizing exon 4 and 2 and these exons do not have target to which they can anneal.