You wish to separate out the following mixture of proteins by size exclusion chromatography under native (non-denaturing) conditions.Protein X is is a homodimer. Each subunit is 25 kDa Protein Y is composed of three subunits that total 120 kDa; two of the subunits are linked by a disulphide bond. Protein Z is a monomer with a molecular weight of 30 kDa. What is their expected order of elution?

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BlackTom AI · Accepted Answer

To reason through the elution order in size exclusion chromatography under native conditions, we consider the apparent molecular sizes of each protein complex as they would be excluded from the gel-like pores.
Option a: Z first, then Y, then X. This would imply Z is the largest species, followed by Y, then X. But Z is only 30 kDa, which is smaller than both Y (120 kDa) and X (50 kDa as a dimer of 25 kDa subunits). Therefore this order cannot be correct.
Option b: Y first, then X, then Z. Here, Y has the largest molecular weight (120 kDa) and, under size exclusion, largest species elute first because they are excluded from the porous network and take a shorter, more direct path. X is a 50 kDa species (a homodimer of 25 kDa subunits), which is intermediate in size, and Z is 30 kDa, the smallest among the three. This order aligns with the general principle of SEC where decreasing size corresponds to later elution, so Y should come out first, followed by X, and then Z.
Option c: X first, then Z, then Y. This would start with the mid-sized X (50 kDa) and then the smallest Z (30 kDa), finishing with the largest Y (120 kDa). This contradicts the principle that larger molecules elute earlier, making this option inconsistent with SEC behavior under native conditions.
Option d: Y first, then Z, then X. While Y is the largest, placing Z before X implies 30 kDa elutes before 50 kDa, which only makes sense if there were an unusual shape or interactions altering the effective size. In standard SEC with globular proteins, molecular weight is the primary determinant of elution order, so this sequence is unlikely.
Overall, the correct conceptual order is based on decreasing apparent size: largest (Y, 120 kDa) first, then intermediate (X, 50 kDa), then smallest (Z, 30 kDa). Therefore, option b best matches the expected elution order in size exclusion chromatography under native conditions.

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