Question

Protein X, MW 15 kDa, pI 4.5

Protein Y, MW 25 kDa, pI 8.5

Protein Z, MW 75 kDa, pI 5.0

In what order would proteins X, Y, and Z elute from a size exclusion column?

Would you expect all three proteins to resolve completely by size exclusion? Briefly, why or why

not?

Answer 1

The size exclusion column chromatography is used to separate molecules based on their size (moelcular weight).

When the given three proteins are passed throgh a gel filteration column (size exclusion column) then the protein with lower mass (protein X) will get adsorbed in the pores of gel (adsorbent), while the protein with higher molecular weight (protein Y and Z) will elute from the column in the order of their moelcular weight. Higher the moelcular weight more the elution(protein Z).

So order will be

First : Z

Then : Y

Then : X

b) all three proteins will not be resolved completely by size exclusion as

(i) the size of polymer and molecular weight are not related similarly for all polymers so we cannot predict the exact elusion

(ii) There is possibility of interaction between polymer and the gel material, hence the polymer will elute later which can be misunderstood to be of smaller size.