You abhor the smell of beta ME and your lab has no DTT Since

You abhor the smell of beta ME and your lab has no DTT. Since there are no disulfide bonds in intracellular proteins (which is predominantly true), you are convinced you do not need to treat your cytosolic samples with mercaptoethanol prior to SDS-PAGE. You do heat your samples in SDS and then run your electrophoresis. Much to your surprise, the gel looks horrible, just one ugly smear (similar to that shown in lane 1 of the figure in part C on page 11). A fellow student suggests you treat your sample with N-ethyl maleimide (NEM), which reacts permanently and irreversibly only with the -SH group of free cysteines (those not involved in disulfide bonds). You run another sample of your cytoplasmic protein mixture after heating it with NEM and SDS. Now the gel (not pictured) looks awesome! If intracellular proteins don\'t have disulfide bonds (which is true), why didn\'t your original scheme work? How does the NEM treatment correct the problem?

Solution

explanation : The reducing environment inside cells keeps disulfide bonds from forming. Once the cellsare broken open and exposed to the atmosphere (oxygen) their environment becomes oxidizing,which allows disulfide bonds to form. This situation is exacerbated during preparation of the samplefor SDS-PAGE.SDS denatures the proteins, which exposes all the sulfhydryl groups. the proteins are applied to the gel in a way that highly concentrates them as they enter the gel,dramatically increasing their opportunities to find inappropriate partners. In addition, the chemicalsused to cross-link the polyacrylamide gel add to the oxidizing environment. NEM prevents formationof inappropriate disulfide bonds by reacting with the free sulfhydryl groups, thereby keeping themout of trouble