Question

There are few questions I wanna ask about the Ion-exchange chromatography.

1. Why we measure the absorbance of the protein fraction at λ₂₈₀ after purification by IEX?

2. After IEX, we are required to carry out BCA assay and L-DOPA assay to determine the protein's concentration and activity but why the instruction said I had to choose the fraction with highest absorbance value?

3. Can you think of one application of IEX to our daily life?

Answer 1

1. The protein contain aromatic amino acids such as tryptophan, tyrosine and phenyl alanine. These aromatic amino acids have phenyl ring which absorbs UV light at 280nm. So purity of protein can be determined by taking absorbance at 280nm. 280nm is a characteristic of purity of protein molecules. For this reason 280nm scan in UV is used in Ion exchange chromatography.

2. Higher absorbtion value means your protein is highly purified. So it is advised to carry out further assay by taking high absorbance value proteins.

3. Ion exchange chromatography can be used in water treatment process where it is used for softening of water and removal of minerals from drinking water