You have a mixture of proteins that you wish to characterize and purify. Here is the information you have on the proteins in your mixture:

You analyze your starting protein mixture by SDS-polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol. After staining, which order of bands would you expect to see on the gel? Each list starts with those at the TOP of the gel and lists the proteins in order going toward the BOTTOM of the gel.

Question

You have a mixture of proteins that you wish to characterize and purify. Here is the information you have on the proteins in your mixture:

You analyze your starting protein mixture by SDS-polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol. After staining, which order of bands would you expect to see on the gel? Each list starts with those at the TOP of the gel and lists the proteins in order going toward the BOTTOM of the gel.

BlackTom AI · Accepted Answer

First, recall that under reducing conditions (β-mercaptoethanol present) disulfide bonds are broken, so any protein that exists as a disulfide-linked multimer will appear as its individual subunits on the SDS-PAGE.
Optionally, note the given total molecular weights (MW): A = 8 kD, B = 90 kD, C = 50 kD, D/E = 42 kD as a dimer (D = 5 kD and E = 37 kD subunits when reduced), and F = 123 kD.
With reduction, the D/E dimer separates into the D (5 kD) and E (37 kD) subunits, so the individual bands to expect are: F (123 kD) at the top, then B (90 kD), then C (50 kD), then E (37 kD), then A (8 kD), then D (5 kD) at the bottom.
Now evaluate each option in light of that predicted order:
- Option 1: F, B, C, D/E, A — this shows the 42 kD D/E as a single unit rather than separated subunits, which is inconsistent with reducing conditions. Also the position of A and D/E misaligns with the expected separate subunits.
- Option 2: D, A, E, C, B, F — this starts with the smallest subunit (D) at the top, which contradicts SDS-PAGE behavior where larger proteins migrate more slowly and appear higher (toward the top). The overall ranking is reversed relative to expectations.
- Option 3: F, B, C, E, A, D — this sequence matches the predicted order: 123 kD (F) at the top, then 90 kD (B), 50 kD (C), 37 kD (E), 8 kD (A), and 5 kD (D) toward the bottom.
- Option 4: E, B, F, D, A, C — this starts with E (37 kD) near the top, which is inconsistent with the largest protein occupying the highest (top) position, and the overall ranking does not align with the expected decreasing MW order.
In summary, the reasoning hinges on the effect of β-mercaptoethanol to reduce disulfide-linked multimers and the well-established principle that, in SDS-PAGE, proteins migrate according to increasing mobility with decreasing molecular weight, placing larger proteins higher up and smaller proteins lower down. The sequence that matches the predicted arrangement after reduction is the one that lists the subunits in the descending MW order: 123, 90, 50, 37, 8, 5.

BIOL3611.13541.202610 InClassQuizMtg8

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