You load the protein mix onto a cation exchange column at pH 5. Next, you apply

Question

You load the protein mix onto a cation exchange column at pH 5. Next, you apply a "washing" step by passing through buffer at pH 5. Finally, for your elution step, you apply a pH gradient starting from pH 2.0 to pH 13.0. (A gradient buffer system allows you to gradually and continuously change the pH of your mobile phase starting from pH 2 up to pH 13).

Indicate the order in which proteins will exit the column during the elution step. Please explain your answer.

Explanation / Answer

In this question , isoelectric points (pl) are giveof all the proteins. As lower the isoelectric points then the proteins becomes more acidic residues and the proteins with more basic residues have high isoelectric point For the elution step we applying pH gradient starting from pH 2.00 to pH 13.00. First we have to look that the pl of the proteins icarries a net positive or negative or zero charge.

If pl value of protein is greater than the pH of buffer, then the protein carries a net positive charge and if pl value of protei is less than the pH of buffer, then the protein carries a net negative charge.

All the proteins which carries a net zero charge will be elute out very first and will not bind to the resin and all the proteins which carry a charge will bnd to the resin and will not elute out easily.

In this question, none of the proteins carries a zero net charge and protein A has least pl and it will elute out first on applying a pH gradient starting from pH 2.00. Then as we gradually increasing the pH gradient of the buffer , other proteins will also elute out in order of their increasing pl value respectively. Thus, the order of eluting of proteins is given as - A>B>D>E>C.

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