A highly-purified protein sample is electrophoresed on a SDS-PAGE gel before and

Question

A highly-purified protein sample is electrophoresed on a SDS-PAGE gel before and after treatment with a high concentration of -mercaptoethanol. Before treatment, the sample exhibited three bands with approcinate molecular weights of 80,000, 65,000 and 40,000. After treatment with -mercaptoethanol, four bands of 80,000, 40,000, 35,000 and 30,000 were detected by electrophoresis. Explain (A): Why does the highly-purified protein sample contain multiple bands on the gel? (B) Why are there four bands after - mercaptoethanol treatment vs. three bands before treatment?

Explanation / Answer

A) The differences in the charge and the different mobility due to different molecular sizes are the two main criteria by which SDS-PAGE separates proteins. If the protein samples contain multiple proteins, multiple protein bands can be obtained on the gel. Even though the protein sample provided in the question is highly-purified, it may contain multiple proteins, which may be the reason for multiple protein bands on the gel.

B) - mercaptoethanol is suitable is a reducing agent for protein disulfide bonds. When the intermolecular disulfide bonds get cleaved, the subunits of a protein gets separated and the can produce individual bands on the gel. This may be the reason for four bands after - mercaptoethanol treatment vs. three bands before treatment.

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