After finishing the purification of a DNA polymerase, researchers were surprised to find by gel filtration two peaks (70kD and 30kD) instead of one. An SDS-PAGE gel of the gel filtration fractions containing the peaks showed that indeed there were two proteins; one was identified as the DNA polymerase (30kD) and the other as a recombinase (40kD). A new purification scheme was devised that isolated the two proteins from each other as shown by gel filtration. The researchers hypothesized the proteins form a complex. The two purified proteins were mixed in a 1:1 ratio and gel filtration was used again. What would be your prediction of the gel filtration chromatography if the hypothesis were incorrect?

Question

After finishing the purification of a DNA polymerase, researchers were surprised to find by gel filtration two peaks (70kD and 30kD) instead of one. An SDS-PAGE gel of the gel filtration fractions containing the peaks showed that indeed there were two proteins; one was identified as the DNA polymerase (30kD) and the other as a recombinase (40kD). A new purification scheme was devised that isolated the two proteins from each other as shown by gel filtration. The researchers hypothesized the proteins form a complex. The two purified proteins were mixed in a 1:1 ratio and gel filtration was used again. What would be your prediction of the gel filtration chromatography if the hypothesis were incorrect?

BlackTom AI · Accepted Answer

Restating the scenario and options to frame the analysis: After purifying two proteins (a 30 kDa DNA polymerase and a 40 kDa recombinase), researchers tested whether they form a complex by mixing them and running gel filtration. If the hypothesis that they form a complex were incorrect, the two proteins would not associate into a larger complex; instead, they would elute as separate species corresponding to their individual molecular weights. The options are:
- only a 40kDa peak
- either a 30kDa or a 40kDa peak
- a 70kDa peak
- only a 30 kDa peak

Option 1: only a 40kDa peak. This would imply that the 30 kDa protein is not present or invisible in the gel filtration after mixing, which is inconsistent with simply mixing two proteins in equal amounts. If both proteins remain separate, you should observe peaks corresponding to both sizes, not just one.

Option 2: either a 30kDa or a 40kDa peak. This option reflects the possibility that the two proteins do not form a stable complex, so each protein could elute independently at its own characteristic volume. In a single run you might observe both peaks, or depending on relative concentrations and detection sensitivity, you might predominantly see one peak; either outcome would be consistent with no complex formation.

Option 3: a 70kDa peak. A 70 kDa peak would suggest the formation of a stable, single complex with combined mass (30 + 40 = 70 kDa). This would support the hypothesis that the proteins form a complex. However, the question asks for the prediction if the hypothesis were incorrect, i.e., if no complex forms, so this option would be inconsistent with that scenario.

Option 4: only a 30 kDa peak. Similar to option 1, this would indicate that only the smaller protein is being detected or eluting, which is not expected if both proteins are present and unbound. If no complex forms, both proteins should be visible, yielding at least one peak at 30 kDa and one at 40 kDa, unless one is undetectable for some reason. In the direct expectation of no complex, this single-peak outcome is unlikely.

Putting it together, the most consistent prediction with the hypothesis being incorrect (i.e., no complex forms) is that you would observe either a 30 kDa peak, or a 40 kDa peak, or both, corresponding to the individual proteins eluting separately. This aligns with the idea that the two proteins do not assemble into a larger single complex under the assay conditions.

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