Question

Please explain what might causes the following results

A. You have bright band on a Northern Blot for a specific RNA, but the Wester blot Shows no band for the protein encoded by that RNA.

B. Your Western blot show that your protein increases size in extracts from cancer cell vs. normal cells.

C. Your Northern blow shows different sized fragments in tissue extracted from brain cells vs heart muscles.

Answer 1

A. Possibility of a nonsense mutation. If some nucleotide within the RNA sequence has undergone nonsense mutation (where a functional amino acid is changed to a stop codon), then the RNA will be intact, but the peptide formed will be prematurely terminated, giving rise to no functional protein. Hence no band in Western blot. Possibility 2: If there is a mutation in the start codon(changing the meaning of the start codon), then translation might not take place at all. So RNA will be there, but no protein will be synthesized. Possibility 3: If there is a frameshift mutation present in the RNA, then a protein of entirely wrong sequence will be synthesized, which will not be recognized by the antibodies used in Western blot. Hence RNA will be there, but bo band in Western blot.

B. In cancer cells, the protein is undergoing some post translational modification like phosphorylation, that is increasing its size. Or the protein in perhaps fused with some other protein in the cancer cells, giving rise to increased size of the protein.

C. Differential splicing of the RNA in brain and heart muscle. The same pre-mRNA can undergo splicing in a different manner in different cells, giving rise to different sized products in the cells. Thus Northern blot will show different sized fragments from the same mRNA in different cells.