Consider the mixture of proteins listed below for questions 10 - 12: You load th

Question

Consider the mixture of proteins listed below for questions 10 - 12: 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

A gradient elution refers to a smooth transition of salt concentration from low to high in the elution buffer. Weakly binding proteins elute first, and stronger binding proteins elute last. If the buffer pH is lower than the pI of the protein, will have strongly positive change. Protein C with pI 10.3 is relatively most positive, so it does not bind well to the resin and elutes first. Protein A will elute last.

Order of elution is:

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