Question

Orange G and Blue dextran are in the mixture. Orange G is a small molecule, whereas blue dextran is relatively big. Which one flows out first? Which one flows out later? Why? 2. You need to purify a protein that is enriched with glutamate (i.e. acidic protein). What kind of chromatography would you use? 3. EGF (epidermal growth factor) is highly associated with malignant breast cancer. You have generated an antibody against EGF to screen the breast tissues. However, there are too many proteins in the sera. How do you isolate EGF from the sera? What chromatography would you use? What would be coated in the column (solid matrix)?

Answer 1

Question-1:

Orange G and Blue Dextran are the two products in the mixture. It is given that Orange G has smaller molecules as it has low molecular weight. Blue Dextran dye has bigger molecules. This mixture is subjected to the size exclusion chromatography using sephadex polymer gel.

First elution ----------------> bigger sized Blue Dextran ----> because it has higher molecular weight

Final elution ----------------> smaller sized Orange G ------> becuase it has smaller molecular weight

Reason: Pricinple of elution by size exclusion chromatography:

1. Size exclusion chromatography or SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis): This technique of purification based on molecular size or molecular weight of the compund.

Princple of size exclusion chromatography if “adsorption”. Larger molecules with more molecular weight cannot pass through pores within the stationary phase (polymer sephadex) and thereby they elute first followed by smaller molecules with low molecular weight as these molecules are bounded in the porous particles of polymer stationary phase.