You have discovered a mutant protein that forms promiscuous disulfide bonds with many other cellular proteins in the endoplasmic reticulum. Using an antibody against the protein, you immunoprecipitate proteins that interact with it. You then separate these proteins using a special type of two-dimensional polyacrylamide-gel electrophoresis (PAGE): in this method, separation in the first dimension is carried out by SDS-PAGE in the absence of β-mercaptoethanol, while separation in the second dimension is carried out by SDS-PAGE in the presence of β-mercaptoethanol. The result is shown in the following schematic drawing. Apparent molecular weights are indicated on the left. What do you think is the molecular weight of the mutant protein? What is the molecular weight represented by the question mark?
A) 15K, 45K
B) 30K, 45K
C) 15K, 30K
D) 30K, 30K
E) 30K, 60K

Question

You have discovered a mutant protein that forms promiscuous disulfide bonds with many other cellular proteins in the endoplasmic reticulum. Using an antibody against the protein, you immunoprecipitate proteins that interact with it. You then separate these proteins using a special type of two-dimensional polyacrylamide-gel electrophoresis (PAGE): in this method, separation in the first dimension is carried out by SDS-PAGE in the absence of β-mercaptoethanol, while separation in the second dimension is carried out by SDS-PAGE in the presence of β-mercaptoethanol. The result is shown in the following schematic drawing. Apparent molecular weights are indicated on the left. What do you think is the molecular weight of the mutant protein? What is the molecular weight represented by the question mark? 
A) 15K, 45K 
B) 30K, 45K 
C) 15K, 30K 
D) 30K, 30K 
E) 30K, 60K

Quizplus · Accepted Answer

All protein complexes separated on this gel presumably contain the mutant protein. Those on the diagonal are associated noncovalently with the mutant protein, and therefore run identically in the two dimensions and end up on the diagonal. The proteins that had formed disulfide bonds with the mutant protein, however, are dissociated from it in the second dimension, resulting in two spots for each such complex, one of which is the common 15K mutant protein. The protein also appears to have dimerized with itself (large spot at the intersection), corresponding to a molecular weight of 30K, indicated by the question mark.

You Have Discovered a Mutant Protein That Forms Promiscuous Disulfide