Question

(3 points) A Student uses cation exchange chromatography to separate Protein 1 pi9.5) and Protein 2 pl 45). The column is equilibrated using a 20m phosphate buffer, pH 6.0 and then the sample containing Protein 1 and Protein 2 is applied to the column. After the sample is applied to the column, the column is first washed with a low salt buffer. Subsequently, the column is washed with a high salt buffer. The elution profile is shown below. Clearly indicate which peak is Protein 1 and which peak is Protein 2. Clearly explain your reasoning. on Change Chromatography perimetere Pro

Answer 1

Protein 1 has isoelectric pH of 9.5 and the elution buffer has 6 .

As , the elution buffer pH is much less than the pi of protein I , then at pH 6 protein will have net positive charge because acid group will not be able to emit H+ and amino group will easily get H+ the net + charge.

Oppositely, protein 2 has pi 4.5 , will have negative charge .

As we have performed cation exchange chromatography, which means that cation will be attracted by negatively charged ions embedded in membrane.

So protein which will have less positivity will come first and which have.more positivity will elute last.

Another way, low salt is needed to elute the low positive protein but high salt concentration is needed to elute the more positive protein.

So I this case ,peak I is for protein 2 and peak ok is for protein 1.

Thank you