Question

Hypothesize the order of elution of the proteins Cytochrome C and Ferritin in both Gel filtration chromatography and ion exchange chromatography with an explanation.

Answer 1

Gel filtration is used to separate proteins such as Cytochrome C and Ferritin of different sizes. It may also determine the native molecular weight of a protein since there is a linear correlation between the elution volume of proteins and the logarithms of their molecular weights (MW).

The separation depends on the ability of molecules to enter the pores.

Smaller molecules like Cytochrome C ( Molecular weight 12 KDa) can diffuse into the beads and move more slowly down the column as compared to the Ferritin (Molecular weight 21 KDa) .

Therefore, Molecules Cytochrome C are eluted in order of decreasing molecular size.

On the other hand ion exchange chromatography (IEC) is the ability to reversibly bind molecules to immobilised charged groups.

Therefore, Elution with a gradient of Cytochrome C and Ferritin. This gradually increases the ionic strength and the molecules are eluted first in Cytochrome C.i.e. the weakly bound molecule which elute first as compared to Ferritin which is strongly bound molecule.

As the solute molecules .i.e. Cytochrome C are released from the column in the order of the strengths of their binding .