Question

You have purified a His-tagged protein expressed in human cell culture by Ni2+ affinity
chromatography.

You have purified a His-tagged protein expressed in human cell culture by Ni^2+ affinity chromatography. When you run the sample on an SDS PAGE gel in the absence of reductant you observe a single band; in the presence of reductant, however, you observe two bands. If you treat with reductant and pour the products over the nickel column, you detect one band in the flow-through and the other band in the eluate. What conclusions can you draw about: primary structure? secondary structure? tertiary structure? quaternary structure? hetero-oligomeric -or- homo-oligomeric complex?

Answer 1

There must be more than one polypeptide.

The primary structure contains cysteines which form disulphide bonds. In the absence of reductant there is a single band. When treated with a reductant, the disulphide bonds are reduced, the polypeptides get separated. The two polypeptides must be of different types, since one band is in the flow through and the other band in the eluate. It can be concluded that the protein is a hetero-oligomer.