You are purifying a protein and performing a Bradford assay after every step of

Question

You are purifying a protein and performing a Bradford assay after every step of purification. First, you put it through anion exchange, and then you put it through size exclusion. After the anion exchange, the concentration of protein was 10 mg/mL. but after size exclusion the protein was only at 5 mg/mL. Assuming we live in a perfect world where the protein is very stable and is not lost due to denaturation (or some other unfortunate occurrence), give an explanation as to why the concentration of protein appears to be lower after the second step.

Explanation / Answer

Obviously, the reason for lower concentration of protein is due to usage of Size Exclusion Chromatography (SEC) in its purification. SEC technique is widely used in the purification of biopolymers such as proteins and DNA. 'SEC' as its name implies works on the principle of elution of different proteins of different sizes on a stationary phase at different rates. Based on this principle SEC is widely used to separate large molecule from smaller molecules.

The separation of analytes in SEC techniques happens because of their passage or non-passage through porous polymeric beads of the column and not because of chemical or electrostatic interactions unlike in other chromatographic separations. As the analyte solution passes through the separation column the smaller molecules pass through the pores of beads of stationary phase while large molecules pass through the space in between the beads. This leads to faster passage of large molecules down the column and leading to their faster elution. Meanwhile the smaller molecules pass through the pores of beads and this delays elution of smaller molecules.

In the example in the question the 50% (wt. /wt.) loss of protein after SEC separation is due to separation of smaller proteins from protein (larger) of interest resulting in the lower amount of desired protein.

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