Given that the protein of interest is a ~50kDa protein, why does Protein A elute

Question

Given that the protein of interest is a ~50kDa protein, why does Protein A elute first in the size exclusion chromatography?

Based on the SDS-PAGE, it can be concluded that Protein B is larger. I thought that in size exclusion chromatography, the larger protein would elute first and smaller proteins elute much slower due to the porous beads in the columns.

Can someone please explain why the sizing column results look like that?

SDS-PAGE:: Sizing Anion Da L Pre: A B A B 10 75 50 25 Anion exchange column: Sizing column: (Elution by salt gradient) Application of 0.75 Elution I Sample 0.50 0.25 Protein A Protein BI 0.00 0.25 Itime 0.00 60.00 AU

Explanation / Answer

Size exclusion chromatography is also known as gel exclusion chromatography or molecular seive. Here, the protein molecules are separated based on their size and shape. In this technique, larger molecules elute first. Elution in SDS-PAGE depends on charge of the molecules. So, protein A being a larger molecule elutes first in the size exclusion chromatography.

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