Question

A scientist subjects the mixture to denaturing Electrophoresis (SDS-PAGE) but forgets to include -mercaptoethanol in the sample buffer. The Coomassie-stained gel has only three bands as seen at right. a) Identify each sample band by labeling with the appropriate letters b)Provide an explanation for i) The number of bands ii) The apparent molecular weight indicated by the band mobilities

Answer 1

Movement of proteins in SDS-PAGE gel is irrespective of pI, because SDS gives equal negative charge and the separation is dependent only on size of polypeptide. But SDS cannot break disulfide bonds. B-mercaptoethanol or DTT can break disulfide bonds and can separate dimers.

1. Top band is for protein A, second band is for the dimer of protein D and third and bottom band is for both protein B and protein C.

1. Out of three bands top one is for protein A, which corresponds to 82000 Da. Second band corresponds to a homodimer of protein D. As mercaptoethanol is not there, dimer was not separated and apparent molecular weight became 44000 Da (22000+22000). The size of the band corresponds to 44000 only. The third bottom band is not resolved and corresponds for protein B and protein C, as they cannot be separated due to smaller difference in their molecular weights 21500 and 23000.