Q7. Which of the two proteins do you expect to elute first from the Sephadex G100 column?

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Question restatement: Q7. Which of the two proteins do you expect to elute first from the Sephadex G100 column?
Options: a) Both are expected to co-elute, b) eGFP first, then Rtms5, c) Rtms5 first, then eGFP

Analysis of each option:

Option a: 'Both are expected to co-elute.' In size-exclusion (Sephadex G100), molecules are separated based on apparent size. Larger molecules are excluded from more pores and thus travel through the column more quickly, while smaller ones enter pores and elute later. Unless the two proteins have nearly identical hydrodynamic radii and there is no overlapping elution range, co-elution is unlikely. Given typical differences in size between a membrane-associated or larger fusion protein (Rtms5) and a smaller soluble fluorescent protein (eGFP), co-elution is not the expected outcome. This option is therefore unlikely.

Option b: 'eGFP first, then Rtms5.' If Rtms5 is the larger protein, it would be expected to elute before the smaller eGFP in size-exclusion chromatography, because the larger protein cannot enter as many pores and thus takes a shorter path through the column. Therefore, predicting eGFP to elute first contradicts the standard behavior of Sephadex G100 for proteins of typical differing sizes. This option contradicts the basic principle of size-exclusion and is incorrect.

Option c: 'Rtms5 first, then eGFP.' This aligns with the general rule for size-exclusion: the larger protein (Rtms5) elutes earlier because it cannot penetrate as many pores and traverses the column more quickly, followed by the smaller protein (eGFP) that enters more pores and elutes later. Assuming Rtms5 is larger in molecular size or hydrodynamic radius than eGFP, this option correctly predicts the elution order.

In sum, option a rests on an unlikely co-elution scenario, option b reverses the expected order given typical size differences, and option c correctly applies the principle of size-exclusion chromatography to predict Rtms5 eluting before eGFP.

BCH2011 S1 2025 Pre-lab 4: MCQ

Q7. Which of the two proteins do you expect to elute first from the Sephadex G100 column?

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